RKEIET 

BRARY 

IVEtSITT  Of 
ILIKMMM 


GY  LIBB. 


STATE  HYGIENIC  LABORATORY 


THE  ELEMENTS 

OF 

BACTERIOLOGICAL 
TECHNIQUE 

A  LABORATORY  GUIDE  FOR 
MEDICAL,  DENTAL,  AND   TECHNICAL    STUDENTS 


BY 


J.   W.   H.^YRE,  M.D.,   M.S.,  F.R.S.    (EoiN.) 

Director  of  the  Bacteriological  Department  of  Guy's  Hospital,  London,  and  Lecturer  on 

Bacteriology  in  the  Medical  and  Dental  Schools  ;  formerly  Lecturer  on  Bacteriology 

at  Charing  Cross  Hospital  Medical  School,  and  Bacteriologist  to  Charing 

Cross  Hospital ;   sometime   Hunterian    Professor,   Royal   College 

of  Surgeons,  England 


SECOND  EDITION 
REWRITTEN  AND  ENLARGED 


PHILADELPHIA  AND   LONDON 

W.   B.   SAUNDERS  COMPANY 

1915 


BIOLOGY  LIBB. 

Copyright,  1902,  by  W.  B.  Saunders  and  Company     Revised,  entirely 
reset,  reprinted,  and  recopyrighted  July,   1913 

Copyright,  1913,  by  W.  B.  Saunders  Company 


Registered  at  Stationers'  Hall,  London,  England 


Reprinted  November,  1915 


PRINTED     IN     AMERICA 


PRESS    OF 

W.     B.    8AUNDERS     COMPANY 
PHILADELPHIA 


£1 

11/3 


BIOLOGY 
LIBRARY 


TO  THE  MEMORY  OF 

JOHN  WICHENFORD  WASHBOURN,  C.M.G.,  M.D.,  F.R.C.P. 

Physician  to  Guy's  Hospital  and  Lecturer  on  Bacteriology  in  the 
Medical  School,  and  Physician  to  the  London  Fever  Hospital 

MY  TEACHER,  FRIEND,  AND  CO-WORKER 


515 


PREFACE  TO  THE  SECOND  EDITION 

BACTERIOLOGY  is  essentially  a  practical  study,  and 
even  the  elements  of  its  technique  can  only  be  taught 
by  personal  instruction  in  the  laboratory.  This  is  a 
self-evident  proposition  that  needs  no  emphasis,  yet 
I  venture  to  believe  that  the  former  collection  of  tried 
and  proved  methods  has  already  been  of  some  utility, 
not  only  to  the  student  in  the  absence  of  his  teacher, 
but  also  to  isolated  workers  in  laboratories  far  removed 
from  centres  of  instruction,  reminding  them  of  for- 
gotten details  in  methods  already  acquired.  If  this 
assumption  is  based  on  fact  no  further  apology  is 
needed  for  the  present  revised  edition  in  which  the 
changes  are  chiefly  in  the  nature  of  additions — ren- 
dered necessary  by  the  introduction  of  new  methods 
during  recent  years. 

I  take  this  opportunity  of  expressing  my  deep 
sense  of  obligation  to  my  confrere  in  the  Physiolog- 
ical Department  of  our  medical  school — Mr.  J.  H. 
Ryffel,  B.  C.,  B.  Sc. — who  has  revised  those  pages 
dealing  with  the  analysis  of  the  metabolic  products 
of  bacterial  life;  to  successive  colleagues  in  the 
Bacteriological  Department  of  Guy's  Hospital,  for 
their  ready  co-operation  in  working  out  or  in  testing 
new  methods;  and  finally  to  my  Chief  Laboratory 
Assistant,  Mr.  J.  C.  Turner  whose  assistance  and  ex- 
perience have  been  of  the  utmost  value  to  me  in  the 
preparation  of  this  volume.  I  have  also  to  thank 
Mrs.  Constant  Ponder  for  many  of  the  new  line 
drawings  and  for  redrawing  a  number  of  the  original 
cuts. 

JOHN  W.  H.  EYRE. 

GUY'S  HOSPITAL,  S.  E. 


PREFACE  TO  THE  FIRST  EDITION 

IN  the  following  pages  I  have  endeavoured  to  ar- 
range briefly  and  concisely  the  various  methods  at 
present  in  use  for  the  study  of  bacteria,  and  the 
elucidation  of  such  points  in  their  life-histories  as  are 
debatable  or  still  undetermined. 

Of  these  methods,  some  are  new,  others  are  not; 
but  all  are  reliable,  only  such  having  been  included 
as  are  capable  of  giving  satisfactory  results  even  in 
the  hands  of  beginners.  In  fact,  the  bulk  of  the 
matter  is  simply  an  elaboration  of  the  typewritten 
notes  distributed  to  some  of  my  laboratory  classes  in 
practical  and  applied  bacteriology;  consequently  an 
attempt  has  been  made  to  present  the  elements  of 
bacteriological  technique  in  their  logical  sequence. 

I  make  no  apology  for  the  space  devoted  to  illus- 
trations, nearly  all  of  which  have  been  prepared 
especially  for  this  volume;  for  a  picture,  if  good, 
possesses  a  higher  educational  value  and  conveys  a 
more  accurate  impression  than  a  page  of  print;  and 
even  sketches  of  apparatus  serve  a  distinct  purpose 
in  suggesting  to  the  student  those  alterations  and 
modifications  which  may  be  rendered  necessary  or 
advisable  by  the  character  of  his  laboratory  equip- 
ment. 

The  excellent  and  appropriate  terminology  intro- 
duced by  Chester  in  his  recent  work  on  "Determina- 
tive Bacteriology"  I  have  adopted  in  its  entirety, 
for  I  consider  it  only  needs  to  be  used  to  convince 
one.  of  its  extreme  utility,  whilst  its  inclusion  in  an 
elementary  manual  is  calculated  to  induce  in  the 
student  habits  of  accurate  observation  and  concise 
description. 

vii 


Vlll  PREFACE    TO    THE    FIRST    EDITION 

With  the  exception  of  Section  XVII— "Outlines 
for  the  Study  of  Pathogenic  Bacteria" — introduced 
with  the  idea  of  completing  the  volume  from  the  point 
of  view  of  the  medical  and  dental  student,  the  work 
has  been  arranged  to  allow  of  its  use  as  a  laboratory 
guide  by  the  technical  student  generally,  whether  of 
brewing,  dairying,  or  agriculture. 

So  alive  am  I  to  its  many  inperfections  that  it 
appears  almost  superfluous  to  state  that  the  book  is 
in  no  sense  intended  as  a  rival  to  the  many  and  ex- 
cellent manuals  of  bacteriology  at  present  in  use, 
but  aims  only  at  supplementing  the  usually  scanty 
details  of  technique,  and  at  instructing  the  student 
how  to  fit  up  and  adapt  apparatus  for  his  daily  work, 
and  how  to  carry  out  thoroughly  and  systematically 
the  various  bacterioscopical  analyses  that  are  daily 
demanded  of  the  bacteriologist  by  the  hygienist. 

Finally,  it  is  with  much  pleasure  that  I  acknowledge 
the  valuable  assistance  received  from  my  late  assistant, 
Mr.  J.  B.  Gall,  A.  I.  C.,  in  the  preparation  of  the  sec- 
tion dealing  with  the  chemical  products  of  bacterial 
life,  and  which  has  been  based  upon  the  work  of 
Lehmann.  JOHN  W.  H.  EYRE. 

GUY'S  HOSPITAL,  S.  E. 


CONTENTS 


PAGE 
I.  LABORATORY  REGULATIONS I 

II.  GLASS  APPARATUS  IN  COMMON  USE 3 

The  Selection,  Preparation,  and  Care  of  Glassware, 
8 — Cleaning  of  Glass  Apparatus,  18 — Plugging 
Test-tubes  and  Flasks,  24. 

III.  METHODS  OF  STERILISATION 26 

Sterilising  Agents,  26 — Methods  of  Application,  27 
— Electric  Signal  Timing  Clock,  38. 

IV.  THE  MICROSCOPE 49 

Essentials,  49 — Accessories,  57 — Methods  of  Micro- 
metry,  61. 

V.  MICROSCOPICAL  EXAMINATION  OF  BACTERIA  AND  OTHER 

MICRO-FUNGI      69 

Apparatus  and  Reagents  used  in  Ordinary  Micro- 
scopical Examination,  69 — Methods  of  Examina- 
tion, 74. 

VI.  STAINING  METHODS 90 

Bacteria  Stains,  90 — Contrast  Stains,  93 — Tissue 
Stains,  95 — Blood  Stains,  97 — Methods  of  Demon- 
strating Structure  of  Bacteria,  99 — Differential 
Methods  of  Staining,  108. 

VII.  METHODS  OF   DEMONSTRATING    BACTERIA   IN  TISSUES  114 
Freezing    Method,    115 — Paraffin    Method,    117 — 
Special  Staining  Methods  for  Sections,  121. 

VIII.  CLASSIFICATION  OF  FUNGI 126 

Morphology  of  the  Hypomycetes,  126 — Morphol- 
ogy of  the  Blastomycetes,  129. 

IX.    SCHIZOMYCETES 13! 

Anatomy,  134 — Physiology,  136 — Biochemistry, 
144. 

X.  NUTRIENT  MEDIA 146 

Meat  Extract,  148 — Standardisation  of  Media,  154 
—The  Filtration  of  Media,  156 — Storing  Media  in 
Bulk,  159 — Tubing  Nutrient  Media,  160. 

XI.  ORDINARY  OR  STOCK  CULTURE  MEDIA 163 

ix 


X  CONTENTS 

PAGE 
XII.  SPECIAL  MEDIA 182 

XIII.  INCUBATORS 216 

XIV.  METHODS  OF  CULTIVATION ".....   221 

Aerobic,  222 — Anaerobic,  236. 

XV.  METHODS  OF  ISOLATION 248 

XVI.  METHODS  OF  IDENTIFICATION  AND  STUDY 259 

Scheme  of  Study,  259 — Macroscopical  Examination 
of  Cultivations,  261 — Microscopical  Methods,  272 — 
Biochemical  Methods,  276 — Physical  Methods,  295 
— Inoculation  Methods,  315 —  Immunisation,  321 — 
Active  Immunisation,  322 — The  Preparation  of 
Hasmolytic  Serum,  327 — The  Titrationof  Haemolytic 
Serum,  328 — Storage  of  Haemolysin,  331. 

XVII.  EXPERIMENTAL  INOCULATION  OF  ANIMALS 332 

Selection  and  Care  of  Animals,  335 — Methods  of 
Inoculation,  352. 

XVIII.  THE  STUDY  OF  EXPERIMENTAL  INFECTIONS  DURING  LIFE  370 
General    Observations,    371 — Blood   Examinations, 
373 — Serological    Investigations,    378 — Agglutinin, 
381 — Opsonin,  387 — Immune  Body,  393. 

XIX.  POST-MORTEM  EXAMINATION  OF  EXPERIMENTAL  ANIMALS  396 
XX.  THE  STUDY  OF  THE  PATHOGENIC  BACTERIA 408 

XXI.  BACTERIOLOGICAL  ANALYSES 415 

Bacteriological  Examination  of  Water,  416 — Ex- 
amination of  Milk,  441 — Ice  Cream,  457 — Ex- 
amination of  Cream  and  Butter,  457 — Examination 
of  Unsound  Meats,  460 — Examination  of  Oysters 
and  Other  Shellfish,  463 — Examination  of  Sewage 
and  Sewage  Effluents,  466 — Examination  of  Air,  468 
— Examination  of  Soil,  470 — Testing  Filters,  478 — 
Testing  of  Disinfectants,  480. 

APPENDIX 492 

INDEX 505 


o  £ 

•3« 


1 


« 


BACTERIOLOGICAL  TECHNIQUE. 

I.  LABORATORY  REGULATIONS. 

The  following  regulations  are  laid  down  for  observ- 
ance in  the  Bacteriological  Laboratories  under  the 
direction  of  the  author.  Similar  regulations  should 
be  enforced  in  all  laboratories  where  pathogenic 
bacteria  are  studied. 


's  Hospital 


BACTERIOLOGICAL    DEPARTMENT. 
HANDLING  OF  INFECTIVE  MATERIALS. 

The  following  Regulations  have  been  drawn  up  in  the  interest 
of  those  working  in  the  Laboratory  as  well  as  the  public  at  large, 
and  will  be  strictly  enforced. 

Their  object  is  to  avoid  the  dangers  of  infection  which  may 
arise  from  neglect  of  necessary  precautions  or  from  carelessness. 

Everyone  must  note  that  by  neglecting  the  general  rules  laid 
down  he  not  only  runs  grave  risk  himself,  but  is  a  danger  to  others. 

REGULATIONS. 

1.  Each  worker  must  wear  a  gown  or  overall,  provided  at  his 
own  expense,  which  must  be  kept  in  the  Laboratory. 

2.  The  hands  must  be  disinfected  with  lysol  2  per  cent,  solu- 
tion, carbolic   acid   5   per  cent,  solution,   or  corrosive   sublimate 
i  per  mille  solution,  after  dealing  with  infectious   material,  and 
before  using  towels. 

3.  On  no  account  must  Laboratory  towels  or  dusters  be  used 
for  wiping  up  infectious  material,  and  if  such  towels  or  dusters  do 
become  soiled,  they  must  be  immediately  sterilised  by  boiling. 

4.  Special  pails  containing  disinfectant  are  provided  to  receive 
any  waste  material,  and  nothing  must  be  thrown  on  the  floor. 

I 


2  LABORATORY   REGULATIONS 

5.  All  instruments  must  be  flamed,  boiled,  or  otherwise  dis- 
infected immediately  after  use. 

6.  Labels  must  be  moistened  with  water,  and  not  by  the  mouth. 

7.  All  disused  cover-glasses,  slides,  and  pipettes  after  use  in 
handling  infectious  material,  etc.,  must  be  placed  in  2  per  cent, 
lysol   solution.     A   vessel    is   supplied   on   each    bench   for   this 
purpose. 

8.  All  plate  and  tube  cultures  of  pathogenic  organisms  when 
done  with,  must  be  placed  for  immediate  disinfection  in  the  boxes 
provided  for  the  purpose. 

9.  No  fluids  are  to  be  discharged  into  sinks  or  drains  unless 
previously  disinfected. 

10.  Animals  are  to  be  dissected  only  after  being  nailed  out  on 
the    wooden    boards,    and    their    skin    thoroughly    washed    with 
disinfectant  solution. 

11.  Immediately  the  post-mortem  examination  is   completed 
each  cadaver  must  be  placed  in  the  zinc  animal-box — without 
removing  the  carcase  from  the  post-mortem  board — and  the  cover 
of  the  box  replaced,  ready  for  carriage  to  the  destructor. 

12.  Dead  animals,  when  done  with,  are  cremated  in  the  destruc- 
tor, and  the  laboratory  attendant  must  be  notified  when  the  bodies 
are  ready  for  cremation. 

13.  None  of  the  workers  in  the  laboratory  are  allowed  to  enter 
the  animal  houses  unless  accompanied  by  the  special  attendant 
in  charge,   who  must  scrupulously  observe  the  same  directions 
regarding  personal  disinfection  as  the  workers  in  the  laboratories. 

14.  No  cultures  are  to  be  taken  out  of  the  laboratory  without 
the  permission  of  the  head  of  the  Department. 

15.  All  accidents,   such  as  spilling  infected  material,    cutting 
or  pricking  the  fingers,  must  be  at  once  reported  to  the  bacteri- 
ologist in  charge. 


II.  GLASS  APPARATUS  IN  COMMON  USE. 

The  equipment  of  the  bacteriological  laboratory,  so 
far  as  the  glass  apparatus  is  concerned,  differs  but 
little  from  that  of  a  chemical  laboratory,  and  the  clean- 
liness of  the  apparatus  is  equally  important.  The 
glassware  comprised  in  the  following  list,  in  addition 
to  being  clean,  must  be  stored  in  a  sterile  or  germ-free 
condition. 

Test=tubes. — It  is  convenient  to  keep  several  sizes 
of  test-tubes  in  stock,  to  meet  special  requirements, 
viz.: 

1.  18x1.5  cm.,  to  contain  media  for  ordinary  tube 
cultivations. 

2.  18X1.3  cm.,  to  contain  media  used  for  pouring 
plate  cultivations,  and  also  for  holding  sterile  "swabs." 

3.  18X2   cm.,  to  contain  wedges  of  potato,   beet- 
root, or  other  vegetable  media. 

4.  13X1.5  cm.,  to  contain  inspissated  blood-serum. 
The  tubes  should  be  made  from  the  best  German 

potash  glass,  "blue-lined,"  stout  and  heavy,  with  the 
edge  of  the  mouth  of  the  tube  slightly  turned  over, 
but  not  to  such  an  extent  as  to  form  a  definite  rim. 
(Cost  about  $1.50,  or  6  shillings  per  gross.)  Such 
tubes  are  expensive  it  is  true,  but  they  are  sufficiently 
stout  to  resist  rough  handling,  do  not  usually  break 
if  accidentally  allowed  to  drop  (a  point  of  some  moment 
when  dealing  with  cultures  of  pathogenic  bacteria) , 
can  be  cleaned,  sterilised,  and  used  over  and  over  again, 
and  by  their  length  of  life  fully  justify  their  initial 
expense. 

A  point  be  noted  is  that  the  manufacturers  rarely 
turn  out  such  tubes  as  these  absolutely  uniform  in 

3 


4  GLASS   APPARATUS    IN   COMMON   USE 

calibre,  and  a  batch  of  1 8  by  1.5  cm.  tubes  usually  con- 
tains such  extreme  sizes  as  1 8  by  2  cm.  and  18  by  1.3 
cm.  Consequently,  if  a  set  of  standard  tubes  is  kept 
for  comparison  or  callipers  are  used  each  new  supply  of 
so-called  18  by  1.5  cm.  tubes  may  be  easily  sorted  out 
into  these  three  sizes,  and  so  simplify  ordering. 

5.  5X0.7  cm.,  for  use  in  the  in  verted  position  inside 
the  tubes  containing  carbohydrate  media,  as  gas- 
collecting  tubes. 

These  tubes,  "unrimmed,"  may  be  of  common  thin 
glass  as  less  than  two  per  cent,  are  fit  for  use  a  second 
time. 


FIG.  i. — Bohemian  flask.     FIG.  2. — Pear-shaped     FIG.  3. — Erlenmeyer  flask 

flask.  (narrow  nock). 

Bohemian  Flasks  (Fig.  i). — These  are  the  ordinary 
flasks  of  the  chemical  laboratory.  A  good  variety, 
ranging  in  capacity  from  250  to  3000  c.c.,  should  be 
kept  on  hand.  A  modified  form,  known  as  the  "  pear- 
shaped"  (Fig.  2),  is  preferable  for  the  smaller  sizes — 
i.  e.j  250  and  500  c.c. 

Erlenmeyer's  Flasks  (Fig.  3). — Erlenmeyer's  flasks 
of  75,  100,  and  250  c.c.  capacity  are  extremely  useful. 
For  use  as  culture  flasks  care  should  be  taken  to  select 
only  such  as  have  a  narrow  neck  of  about  2  cm.  in 
length. 

Kolle's  Culture  Flasks  (Fig.  4).— These  thin,  flat 
flasks  (to  contain  agar  or  gelatine,  which  is  allowed  to 
solidify  in  a  layer  on  one  side)  are  extremely  useful 


KOLLE'S  CULTURE  FLASKS  5 

on  account  of  the  large  nutrient  surface  available  for 
growth.  A  surface  cultivation  in  one  of  these  will 
yield  as  much  growth  as  ten  or  twelve  "oblique"  tube 
cultures.  The  wide  mouth,  however,  is  a  disadvantage, 


FIG.  4. — Kolle's  cul- 
ture flask. 


FIG.  5. — Roux's 
culture  bottle. 


FIG.  6. — Guy's  culture 
bottle. 


and  for  many  purposes  thin,  flat  culture  bottles  known 
as  Roux's  bottles  (Fig.  5)  are  to  be  preferred. 

An  even  more  convenient  pattern  is  that  used  in  the 
author's  laboratory  (Fig.  6),  as  owing  to  the  greater 


FIG.  7.— Filter  flask. 

depth  of  medium  which  it  is  possible  .to  obtain  in  these 
flasks  an  exceedingly  luxuriant  growth  is  possible ;  the 
narrow  neck  reduces  the  chance  of  accidental  contami- 
nation to  a  minimum  and  the  general  shape  permits 
the  flasks  to  be  stacked  one  upon  the  other. 


6  GLASS   APPARATUS    IN   COMMON    USE 

Filter  Flasks  or  Kitasato's  Serum  Flasks  (Fig.  7).— 
Various  sizes,  from  250  to  2000  c.c.  capacity.  These 
must  be  of  stout  glass,  to  resist  the  pressure  to  which 
they  are  subjected,  but  at  the  same  time  must  be 
thoroughly  well  annealed,  in  order  to  withstand  the 
temperature  necessary  for  sterilisation. 

All  flasks  should  be  either  of  Jena  glass  or  the  almost 
equally  well-known  Resistance  or  R  glass,  the  extra 
initial  expense  being  justified  by  the  comparative 
immunity  of  the  glass  from  breakage. 

Petri's  Dishes  or  "Plates"  (Fig.  8,  a).— These  have 
now  completely  replaced  the  rectangular  sheets  of  glass 
introduced  by  Koch  for  the  plate  method  of  cultiva- 
tion. Each  "plate"  consists  of  a  pair  of  circular  discs 
of  glass  with  sharply  upturned  edges,  thus  forming 
shallow  dishes,  one  of  slightly  greater  diameter  than 
the  other,  and  so,  when  inverted,  forming  a  cover  or 
cap  for  the  smaller.  Plates  having  an  outside  diam- 
eter of  10  cm.  and  a  height  of  1.5  cm.  are  the  most 
generally  useful.  A  batch  of  eighteen  such  plates  is 
sterilised  and  stored  in  a  cylindrical  copper  box  (30 
cm.  high  by  12  cm.  diameter)  provided  with  a  "  pull-off" 
lid.  Inside  each  box  is  a  copper  stirrup  with  a  circular 
bottom,  upon  which  the  plates  rest,  and  by  means  of 
which  each  can  be  raised  in  turn  to  the  mouth  of  the  box 
(Fig.  9)  for  removal. 

Capsules  (Fig.  8,  b  and  c). — These  are  Petri's  dishes 
of  smaller  diameter  but  greater  depth  than  those  termed 
plates.  Two  sizes  will  be  found  especially  useful — 
viz.,  4  cm.  diameter  by  2  cm.  high,  capacity  about  14 
c.c. ;  and  5  cm.  diameter  by  2  cm.  high,  capacity 
about  25  c.c.  These  are  stored  in  copper  cylinders  of 
similar  construction  to  those  used  for  plates,  but 
measuring  20  by  6  cm.  and  20  by  7  cm.,  respectively. 

Graduated  Pipettes. — Several  varieties  of  these  are 
required,  viz.: 

i.  Pipettes  of  i  c.c.  capacity  graduated  in  o.i  c.c. 


GRADUATED    PIPETTES 


2.  Pipettes  of  i  c.c.  capacity  graduated  in  o.oi  c.c. 
(Fig.  10,  a). 


FIG.  8.— Petri  dish  (a),  and 
capsules  (b,  c). 


FIG.  9. — Plate  box 
with  stirrup. 


3.  Pipettes  of  10  c.c.  capacity  graduated  in  o.i  c.c. 
(Fig.   10,  b). 

These    should   be   about   30   cm.   in     a  b 

length  (i  and  2  of  fairly  narrow  bore) , 
graduated  to  the  extreme  point,  and 
having  at  least  a  10  cm.  length  of  clear 
space  between  the  first  graduation  and 
the  upper  end ;  the  open  mouth  should  be 
plugged  with  cotton- wool.  Each  variety 
should  be  sterilised  and  stored  in  a 
separate  cylindrical  copper  case  some 
36  by  6  cm.,  with  "pull-off"  lid,  upon 
which  is  stamped,  in  plain  figures,  the 
capacity  of  the  contained  pipettes. 

The  laboratory  should  also  be  pro- 
vided with  a  complete  set  of  ' '  Standard  "  FIG  .  i  o .  — 
graduated  pipettes,  each  pipette  in  the 
set  being  stamped  and  authenticated  by 
a  certificate  from  one  of  the  recognised  Physical 
Measurement  Laboratories,  such  as  Charlottenburg. 


8 


GLASS   APPARATUS   IN   COMMON   USE 


These  instruments  are  expensive  and  should  be  reserved 
solely  for  standardising  the  pipettes  in  ordinary  use,  and 
for  calibrating  small  pipettes  manufactured  in  the 
laboratory.  Such  a  set  should  comprise,  at  least, 
pipettes  delivering  10  c.c.,  5  c.c.,  2.5  c.c.,  2  c.c.,  i  c.c., 
0.5  c.c.,  0.25  c.c. ,  0.2  c.c.,  o.i  c.c.,  0.05  c.  c.,  and  o.oi  c.c., 
respectively. 

In  the  immediately  following  sections  are  described 
small  pieces  of  glass  apparatus  which  should  be  prepared 
in  the  laboratory  from  glass  tubing  of  various  sizes. 
In  their  preparation  three  articles  are  essential;  first 
a  three-square  hard-steel  file  or  preferably  a  glass- work- 
er's knife  of  hard  Thuringian  steel  for  cutting  glass  tubes 
etc. ;  next  a  blowpipe  flame,  for  although  much  can  be 
done  with  the  ordinary  Bunsen  burner,  a  blowpipe 
flame  makes  for  rapid  work;  and  lastly  a  bat's- wing 
burner. 

i .  The  glass-cutting  knife.  This  article  is  sold  in  two 
forms,  a  bench  knife  (Fig.  1 1)  and  a  pocket  knife.  The 
former  is  provided  with  a  blade  some  8  cm.  in  length 


FIG.  ii. — Glass-cutting  knife,     a.  handle.     6,  double  edged  blade. 
c.  shaft     d.  locking  nut.     e.  spanner  for  nut. 


and  having  two  cutting  edges.  The  cutting  edge  when 
examined  in  a  strong  light  is  seen  to  be  composed  of  small 
closely  set  teeth,  similar  to  those  in  a  saw.  The  knife 
should  be  kept  sharp  by  frequent  strappings  on  a 
sandstone  hone.  The  pocket  form,  about  6-cm.  long 


SEDIMENTATION    TUBES 


over  all,  consists  of  a  small  spring  blade  with  one 
cutting  edge  mounted  in  scales  like  an  ordinary  pocket 
knife. 

2.  For  real  convenience  of  work  the  blowpipe  should 
be  mounted  on   a   special  table  connected   up   with 
cylindrical  bellows  operated  by  a  pedal.     That  figured 
(Fig.  12)  is  made  by  mounting  a  teak  top  60  cm.  square 
upon  the  uprights  of  an  enclosed  double-action  concer- 
tina bellows  (Enfer's)  and  provided  with  a  Fletcher's 
Universal  gas  blowpipe. 

3.  An  ordinary  bat's- wing  gas-burner  mounted  at  the 
far  corner  of  the  table  top  is  invaluable  in  the  prepara- 
tion of  tubular  apparatus  with  sharp  curves,  and  for 
coating  newly-made  glass  apparatus  with  a  layer  of 
soot  to  prevent  too  rapid  cooling,  and  its  usually  asso- 
ciated result — cracking. 

6.  Sedimentation  tubes  5x0.5.  cm.,  for  sedimentation 
reactions,  etc.,  and  for  containing  small  quantities  of 
fluid  to  be  centrifugalisde  in  the  hsematocrit.  These 
are  made  by  taking  14-011.  lengths  of  stout  glass  tubing 


FIG.  12. — Glass  blower's  table  with  Enfer's  foot  bellows. 

of  the  requisite  diameter  and  heating  the  centre  in  the 
Bunsen  or  blow-pipe  flame.  When  the  central  portion 
is  quite  soft  draw  the  ends  quickly  apart  and  then 
round  off  the  pointed  ends  of  the  two  test-tubes  thus 


10 


GLASS  APPARATUS   IN   COMMON   USE 


formed.  With  the  glass-cutting  knife  cut  off  whatever 
may  be  necessary  from  the  open  ends  to  make  the 
tubes  the  required  length. 

A  rectangular  block  of  "plasticine"  (modelling  clay) 
into  which  the  conical  ends  can  be  thrust  makes  a 
very  convenient  stand  for  these  small  tubes. 

Capillary  Pipettes  or  Pasteur's  Pipettes  (Fig.  13  a). — 
These  little  instruments  are  invaluable,  and  a  goodly 
a.  b.  c.  supply  should  be  kept  on  hand.  They 
are  prepared  from  soft-glass  tubing  of 
various-sized  calibre  (the  most  generally 
useful  size  being  8  mm. diameter)  in  the 
following  manner :  Hold  a  10  cm.  length 
of  glass  tube  by  each  end,  and  whilst 
rotating  it  heat  the  central  portion  in 
the  Bunsen  flame  or  the  blow-pipe 
blast-flame  until  the  glass  is  red  hot 
and  soft.  Now  remove  it  from  the  flame 
and  steadily  pull  the  ends  apart,  so 
drawing  the  heated  portion  out  into  a 
roomy  capillary  tube;  break  the  capil- 
lary portion  at  its  centre,  seal  the 
broken  ends  in  the  flame,  and  round 
off  the  edges  of  the  open  end  of  each 
pipette.  A  loose  plug  of  cotton-wool 
in  the  open  mouth  completes  the  capil- 
lary pipette.  After  a  number  have 
been  prepared,  they  are  sterilised  and 
.  stored  in  batches,  either  in  metal  cases 

FIG.    13. — Capil- 
lary pipettes.       a,  similar  to  those  used  for  the  graduated 

pipettes  or  in  large-sized  test-tubes — 
sealed  ends  downward  and  plugged  ends  toward  the 
mouth  of  the  case. 

The  filling  and  emptying  of  the  capillary  pipette 
is  most  satisfactorily  accomplished  by  slipping  a  small 
rubber  teat  (similar  to  that  on  a  baby's  feeding  bottle 
but  not  perforated)  on  the  upper  end,  after  cutting  or 


BLOOD    PIPETTES  II 

snapping  off  the  sealed  point  of  the  capillary  portion. 
If  pressure  is  now  exerted  upon  the  elastic  bulb  by  a 
ringer  and  thumb  whilst  the  capillary  end  is  below  the 
surface  of  the  fluid  to  be  taken  up,  some  of  the 
contained  air  will  be  driven  out,  and  subsequent  relax- 
ation of  that  pressure  (resulting  in  the  formation  of  a 
partial  vacuum)  will  cause  the  fluid  to  ascend  the 
capillary  tube.  Subsequent  compression  of  the  bulb 
will  naturally  result  in  the  complete  expulsion  of  the 
fluid  from  the  pipette  (Fig.  14). 


FIG.   14. — Filling  the  capillary  teat-pipette. 

A  modification  of  this  pipette,  in  which  a  constric- 
tion or  short  length  of  capillary  tube  is  introduced 
just  below  the  plugged  mouth  (Fig.  13,  6),  will  also  be 
found  extremely  useful  in  the  collection  and  storage 
of  morbid  exudations. 

A  third  form,  where  the  capillary  portion  is  about 
4  or  5  cm.  long  and  only  forms  a  small  fraction  of  the 
entire  length  of  the  pipette  (Fig.  13,  c),  will  also  be 
found  useful. 

"Blood"  Pipettes  (Fig  15). — Special  pipettes  for 
the  collection  of  fairly  large  quantities  of  blood  (as 
suggested  by  Pakes)  should  also  be  prepared.  These 
are  made  from  soft  glass  tubing  of  i  cm.  bore,  in  a 
similar  manner  to  the  Pasteur  pipettes,  except  that 


12 


GLASS   APPARATUS   IN   COMMON    USE 


the  point  of  the  blowpipe  flame  must  be  used  in  order 
to  obtain  the  sharp  shoulder  at  either  end  of  the  cen- 
tral bulb.  The  terminal  tubes  must  retain  a  diameter 
of  at  least  i  mm.,  in  order  to  avoid  capillary  action 
during  the  collection  of  the  fluid. 

For  sterilisation  and  storage  each  pipette  is  placed 
inside  a  test-tube,  resting  on  a  wad  of  cotton-wool,  and 
the  tube  plugged  in  the  ordinary  manner.  As  these 
tubes  are  used  almost  exclusively  for  blood  work, 


FIG.  15. — Blood  pipettes 
and  hair-lip  pin  in  a  test- 
tube. 


•_j=» 

FIG.  16. — Blood-pipette  in  metal 
thermometer  case. 


it  is  usual  to  place  a  lance-headed  hare-lip  pin  or  a 
No.  9  flat  Hagedorn  needle  inside  the  tube  so  that  the 
entire  outfit  may  be  sterilised  at  one  time. 

For  the  collection  of  small  quantities  of  blood  for 
agglutination  reactions  and  the  like,  many  prefer  a 
short  straight  piece  of  narrow  glass  tubing  drawn  out 
at  either  extremity  to  almost  capillary  dimensions. 
Such  pipettes,  about  8  cm.  in  length  over  all,  are  most 


AUTOMATIC    PIPETTES  13 

conveniently  sterilized  in  ordinary  metal  thermometer 
cases  (Fig.  16). 

Graduated  Capillary  Pipettes  (Fig.  17). — These  should 
also  be  made  in  the  laboratory — from  manometer 
tubing — of  simple,  convenient  shape,  and  graduated 
by  the  aid  of  " standard"  pipettes  (in  hundredths)  to 
contain  such  quantities  as  10,  50,  and 
90  c.mm.,  and  carefully  marked  with  a 
writing  diamond.  These,  previously 
sterilised  in  large  test-tubes,  will  be 
found  extremely  useful  in  preparing 
accurate  percentage  solutions,  when 
only  minute  quantities  of  fluid  are 
available. 

Automatic  ("Throttle")  Pipettes.— 
These  ingenious  pipettes,  introduced 
by  Wright,  can  easily  be  calibrated 
in  the  laboratory  and  are  exceedingly 
useful  for  graduating  small  pipettes,  FlG.  I7.— capii- 
for  measuring  small  quantities  of  fluids,  l?*y  graduated 

1M    A.  -  ,        pipettes. 

in  prepanng  dilutions  of  serum  tor 
agglutination  reactions,  etc.  They  are  usually  made 
from  the  Capillary  Pasteur  pipettes  (Fig.  13,  a).  The 
following  description  of  the  manufacture  of  a  5  c.mm. 
pipette  will  serve  to  show  how  the  small  automatic 
pipettes  are  calibrated. 

1.  Select  a  pipette  the  capillary  portion  of  which  is 
fairly  roomy  in  bore  and  possesses  regular  even  walls, 
and  remove  the  cotton- wool  plug  from  the  open  end. 

2 .  Heat  the  capillary  portion  near  the  free  extremity 
in  the  by-pass  flame  of  the  bunsen  burner  and  draw  it 
out  into  a  very  fine  hair-like  tube  and  break  this 
across.     This  hair-like  extremity  will  permit  the  pas- 
sage of  air  but  is  too  fine  for  metallic  mercury  to  pass. 

3.  From    a    standard    graduated    pipette    deliver 
5  c.mm.  clean  mercury  into  the  upper  wide  portion  of 
the  pipette. 


14  GLASS  APPARATUS   IN   COMMON   USE 

4.  Adjust  a  rubber  teat  to  the  pipette  and  by  pres- 
sure on  the  bulb  gradually  drive  the  mercury  in  an 
unbroken  column  down  the  capillary  tube   until  it  is 
stopped  by  the  filiform  extremity. 

5.  Cut  off  the  capillary  tube  exactly  at  the  upper 
level  of  the  column  of  mercury,  invert  it  and  allow 
the  mercury  to  run  out. 

6.  Snap  off  the  remainder  of  the  capillary  tube  from 
the  broad  upper  portion  of  the  pipette  which  is  now 

destined  to  form  the  covering  tube  or 
air  chamber,  or  what  we  may  term  the 
"  barrel."  This  barrel  now  has  the  lower 
end  in  the  form  of  a  truncated  cone,  the 
upper  end  being  cut  square.  Remove 
the  teat. 

7.  Introduce   the  capillary  tube  into 
this    barrel  with  the  filiform  extremity 
uppermost,  and  the  square  cut  end  pro- 
jecting about  0.5  cm.  beyond  the  taper- 
ing end  of  the  barrel. 

8.  Drop  a  small  pellet  of  sealing  wax 
into  the  barrel  by  the  side  of  the  capil- 
lary tube  and  then  warm    the  tube  at 

FIG.     1 8.—  the    gas    flame  until    the  wax  becomes 
Throttle  pipette  softened  and  makes  an   air-tight    joint 

— small  capacity. 

between  the  capillary  tube  and  the  end 
of  the  barrel. 

9.  Fit  a  rubber  teat  to  the  open  end  of  the  barrel, 
and  so  complete  a  pipette  which  can  be  depended  upon 
to  always  aspirate  and  deliver  exactly  5  cm.  of  fluid. 

Slight  modification  of  this  procedure  is  necessary  in 
making  tubes  to  measure  larger  volumes  than  say 
75  c.mm.  Thus  to  make  a  throttle  pipette  to  measure 
100  c.mm.: 

i.  Take  a  short  length  of  quill  tubing  and  draw  out 
one  end  into  a  roomy  capillary  stem,  and  again  draw 
out  the  extremity  into  a  fine  hair  point,  thus  forming 


AUTOMATIC  PIPETTES 


a   small   Pasteur   pipette   with   a   hair-like    capillary 
extremity. 

2.  With  a  standard  pipette  fill  100  c.mm.  into  the 
neck  of  this  pipette,  and  make  a  scratch  with  a  writing 
diamond  at  the  upper  level  (a)  of  the  mercury  meniscus 
(Fig.  1 9,  A). 


FIG.  19. — Making  throttle  pipettes — large  capacity 

Now  force  the  mercury  down  into  the  capillary  stem 
as  far  as  it  will  go,  so  as  to  leave  the  upper  part  of  the 
tube  in  the  region  of  the  diamond  scratch  empty 
(Fig.  19,  B). 

3 .  Heat  the  tube  in  the  region  of  the  diamond  scratch 
in  the  blowpipe  flame,  and  removing  the  tube  from 
the  flame  draw  it  out  so  that  the  diamond  scratch  now 
occupies  a  position  somewhere  near  the  centre  of  this 
new  capillary  portion  (Fig.  19,  C). 


1 6  GLASS   APPARATUS   IN   COMMON   USE 

4.  Heat  the  tube  in  this  position  in  the  peep  flame 
of  the  Bunsen  burner,  and  draw  it  out  into  a  hair-like 
extremity.     Snap   off  the  glass  tube,   leaving  about 
5  mm.  of  hair-like  extremity  attached  to  the  upper 
capillary  portion  (Fig.  19,  D) .     Allow  the  glass  to  cool. 

5.  Lift  up  the  bulb  by  the  long  capillary  stem  and 
allow  the  mercury  to  return  to  its  original  position — 
an   operation  which  will  be  facilitated  by  snapping 
off   the   hair-like   extremity  from    the  long   piece  of 
capillary  tubing. 

6.  Mark  on  the  capillary  stem  with  a  grease  pencil 
the  position  of  the  end  of   the  column  of   mercury 
(Pig.  19,  E.) 

7.  Warm  the   capillary  tubing  at  this  spot  in  the 
peep  flame  of  the  Bunsen  burner,  and  draw  it  out  very 
slightly  so  that  when  cut  at  this  position  a  pointed 
extremity  will  be  obtained. 

8.  With  a  glass-cutting  knife  cut  the  capillary  tube 
through  at  the  point  "6,"  and  allow  the  mercury  to 
run  out. 

9.  Now  apply  a  thick  layer  of  sealing  wax  to  the 
neck  of  the  bulb. 

10.  Take  a  piece  of  5  mm.  bore  glass  tubing  and 
draw  it  out  as  if  making  an  ordinary  Pasteur  pipette. 

11.  Break  the  capillary  portion  off  so  as  to  leave  a 
covering  tube  similar  to  that  already  used  for  the 
smaller  graduated  pipettes.     Into  this  covering  tube 
drop  the  graduated  bulb  and  draw  the  capillary  stem 
down    through    the    conical    extremity    until    further 
progress  is  stopped  by  the  layer  of  sealing  wax. 

12.  Warm  the  pipette  in  the  gas  flame  so  as  to  melt 
the  sealing  wax  and  make  an  air-tight  joint. 

13.  Fit  an  india-rubber  teat  over  the  open  end  of  the 
covering  tube,  and  the  automatic  pipette  is  ready  for 
use  (Fig.  19,  F). 

Sedimentation  Pipettes  (Fig.  20) . — These  are  prepared 
from  10  cm.  lengths  of  narrow  glass  tubing  by  sealing 


FERMENTATION    TUBES 


one  extremity,  blowing  a  small  bulb  at  the  centre,  and 
plugging  the  open  end  with  cotton-wool ;  after  sterilisa- 
tion the  open  end  is  provided  with  a  short  piece  of 
rubber  tubing  and  a  glass  mouthpiece.  When  it  is 
necessary  to  observe  sedimentation  reactions  in  very 
small  quantities  of  fluid,  these  tubes  will  be  found 


FIG.  20. — Sedimentation  pipette. 

much  more  convenient  than  the  5  by  0.5  cm.  test-tubes 
previously  mentioned. 

Pasteur  pipettes  fitted  with  india-rubber  teats  will 
also  be  found  useful  for  sedimentation  tests  when 
dealing  with  minute  quantities  of  serum,  etc. 

Fermentation  Tubes  (Fig.  21). — These  are  used  for 
the  collection  and  analysis  of  the  gases  liberated  from 


b  c 

FIG.  21. — Fermentation  tubes. 

the  media  during  the  growth  of  some  varieties  of  bac- 
teria and  may  be  either  plain  (a)  or  graduated  (b). 
A  simple  form  (Fig.  21,  c)  may  be  made  from  14 
cm.  lengths  of  soft  glass  tubing  of  1.5  cm.  diameter. 
The  Bunsen  flame  is  applied  to  a  spot  some  5  cm.  from 
one  end  of  such  a  piece  of  tubing  and  the  tube  slightly 
drawn  out  to  form  a  constriction,  the  constricted  part 


1 8  GLASS   APPARATUS    IN   COMMON    USE 

is  bent  in  the  bat's- wing  flame,  to  an  acute  angle,  and 
the  open  extremity  of  the  long  arm  sealed  off  in  the 
blowpipe  flame.  The  open  end  of  the  short  arm  is 
rounded  off  and  then  plugged  with  cotton-wool,  and 
the  tube  is  ready  for  sterilisation. 

CLEANING  OF  GLASS  APPARATUS. 

All  glassware  used  in  the  bacteriological  laboratory 
must  be  thoroughly  cleaned  before  use,  and  this  rule 
applies  as  forcibly  to  new  as  to  old  apparatus,  although 
the  methods  employed  may  vary  slightly. 

To  Clean  New  Test=tubes.— 

1.  Place  the  tubes  in  a  bucket  or  other  convenient 
receptacle,  fill  with  water  and  add  a  handful  of  "  Sapon  " 
or  other  soap  powder.     See  that  the  tubes  are  full  and 
submerged. 

2.  Fix  the  bucket  over  a  large  Bunsen  flame  and  boil 
for  thirty  minutes — or   boil  in   the   autoclave  for  a 
similar  period. 

3.  Cleanse  the  interior  of  the  tubes  with  the  aid  of 
test-tube  brushes,  and  rinse  thoroughly  in  cold  water. 

4.  Invert  the  tubes  and  allow  them  to  drain  com- 
pletely. 

5.  Dry  the  tubes  and  polish  the  glass  inside  and  out 
with  a  soft  cloth,  such  as  selvyt. 

New  flasks,  plates,  and  capsules  must  be  cleaned  in 
a  similar  manner. 

To  Clean  New  Graduated  Pipettes. — 

1.  Place  the  pipettes  in  a  convenient   receptacle, 
filled  with  water  to  which  soap  powder  has  been  added. 

2.  Boil  the  water  vigorously  for  twenty  minutes  over 
a  Bunsen  flame. 

3.  Rinse  the  pipettes  in  running  water  and  drain. 

4.  Run  distilled  water  through  the  pipettes  and 
drain. 


CLEANING    INFECTED    TEST-TUBES  19 

5.  Run  rectified  spirits  through  the  pipette  and  drain 
as  completely  as  possible. 

6.  Place  the  pipettes  in  the  hot-air  oven'  (vide  page 
31),  close  the  door,  open  the  ventilating  slide,  and 
run  the  temperature  slowly  up  to  about  80°  C.     Turn 
off  the  gas  and  allow  the  oven  to  cool. 

Or  6a.  Attach  each  pipette  in  turn  to  the  rubber 
tube  of  the  foot  bellows,  or  blowpipe  air-blast,  and 
blow  air  through  the  pipette  until  the  interior  is  dry. 

Glassware  that  has  already  been  used  is  regarded  as 
infected,  and  is  treated  in  a  slightly  different  manner. 

Infected  Test=tubes.— 

1.  Pack  the  tubes  in  the  wire  basket  of  the  auto- 
clave   (having    previously    removed    the    cotton-wool 
plugs,  caps,  etc.),  in  the  vertical  position,  and  before 
replacing  the  basket  see  that  there  is  a  sufficiency  of 
water  in  the  bottom  of  the  boiler.     Now  attach  a 
piece  of  rubber  tubing  to  the  nearest  water  tap,  and 
by  means  of  this  fill  each  tube  with  water. 

2.  Disinfect  completely  by  exposing  the  tubes,  etc., 
to  a  temperature  of  120°  C.  for  twenty  minutes  (vide 

page  37). 

(If  an  autoclave  is  not  available,  the  tubes  must  be 
placed  in  a  digester,  or  even  a  large  pan  or  pail  with  a 
tightly  fitting  cover,  and  boiled  vigorously  for  some 
thirty  to  forty-five  minutes  to  ensure  disinfection.) 

3.  Whilst  still  hot,  empty  each  tube  in  turn  and 
roughly  clean  its  interior  with  a  stiff  test-tube  brush. 

4.  Place  the  tubes  in  a  bucket  or  other  convenient 
receptacle,  fill  with  water  and  add  a  handful  of  Sapon 
or  other  soap  powder.     See  that  the  tubes  are  full  and 
submerged. 

5.  Fix  the  bucket  over  a  large  Bunsen  flame  and 
boil  for  thirty  minutes. 

6.  Cleanse  the  interior  of  the  tubes  with  the  aid  of 
test-tube  brushes,  and  rinse  thoroughly  in  cold  water. 


2O  GLASS   APPARATUS   IN   COMMON   USE 

7.  Drain  off  the  water  and  immerse  tubes  in  a  large 
jar  containing  water  acidulated  with  2  to  5  per  cent, 
hydrochloric  acid.     Allow  them  to  remain  there  for 
about  fifteen  minutes. 

8.  Remove  from  the  acid  jar,  drain,  rinse  thoroughly 
in  running  water,  then  with  distilled  water. 

9.  Invert  the  tubes  and  allow  them  to  drain  com- 
pletely. 

Dry  the  tubes  and  polish  the  glass  inside  and  out 
with  a  soft  cloth,  such  as  selvyt. 

Infected  flasks,  plates,  and  capsules  must  be  treated 
in  a  similar  manner. 

Flasks  which  have  been  used  only  in  the  preparation 
of  media  must  be  cleaned  immediately  they  are  finished 
with.  Fill  each  flask  with  water  to  which  some  soap 
powder  and  a  few  crystals  of  potassium  permanganate 
have  been  added,  and  let  boil  over  the  naked  flame. 
The  interior  of  the  flask  can  then  usually  be  perfectly 
cleaned  with  the  aid  of  a  flask  brush,  but  in  some  cases 
water  acidulated  with  5  per  cent,  nitric  acid,  or  a  large 
wad  of  wet  cotton-wool  previously  rolled  in  silver  sand, 
must  be  shaken  around  the  interior  of  the  flask,  after 
which  rinse  thoroughly  with  clean  water,  dry,  and 
polish. 

Infected  Pipettes.— 

1.  Plunge  infected  pipettes  immediately  after  use 
into  tall  glass  cylinders  containing  a  2  per  cent,  solu- 
tion of  lysol,  and  allow  them  to  remain  therein  for 
some  days. 

2.  Remove  from  the  jar  and  drain.     Boil  in  water 
to   which   a   little   soap   has   been   added,  for  thirty 
minutes. 

3.  Rinse  thoroughly  in  cold  water. 

4.  Immerse  in  5  per  cent,  nitric  acid  for  an  hour  or 
two. 


CLEANING    PIPETTES 


21 


5.  Rinse  again  in  running  water  to  remove  all  traces 
of  acid. 

6.  Complete  the  cleaning  as  described  under  "new 
pipettes." 

When  dealing  with  graduated  capillary  pipettes  em- 
ployed for  blood  or  serum  work  (whether  new  or  in- 
fected), much  time  is  consumed  in  the  various  steps 
from  5  onward,  and  the  cleansing  process  can  be  mate- 
rially hastened  if  the  following  device  is  adopted. 

Fit  up  a  large-sized  Kitasato's  filter  flask  to  a  Spren- 
gel's  suction  pump  or  a  Geryk  air  pump  (see  page 
43).  To  the  side  tubulure  of  the  filter  flask  attach 
a  20  cm.  length  of  rubber  pressure  tubing  having 
a  calibre  sufficiently  large  to  admit  the  ends  of  the 
pipettes. 

Next  fill  a  small  beaker  with  distilled  water.  Attach 
the  first  pipette  to  the  free  end  of  the  rubber  tubing, 


FIG.  22. — Cleaning  blood  pipettes. 

place  the  pipette  point  downward  in  the  beaker  of 
water  and  start  the  pump  (Fig.  22). 

When  all  the  water  has  been  aspirated  through  the 
pipette  into  the  filter  flask,  fill  the  beaker  with  recti- 
fied spirit  and  when  this  is  exhausted  refill  with  ether. 
Detach  the  pipette  and  dry  in  the  hot-air  oven. 

Slides  and  cover  =slips  (Fig.  23),  when  first  purchased, 


22 


GLASS  APPARATUS   IN   COMMON    USE 


have  " greasy"  surfaces,  upon  which  water  gathers  in 
minute  drops  and  effectually  prevents  the  spreading 
of  thin,  even  films. 

Microscopical  Slides. — The  slides  in  general  use  are 
those  known  as  " three  by  one"  slips  (measuring  3 
inches  by  i  inch,  or  76  by  26  mm.),  and  should  be 
of  good  white  crown  glass,  with  ground  edges. 

New  slides  should  be  allowed  to  remain  in  alcohol 
acidulated  with  5  per  cent,  hydrochloric  acid  for  some 
hours,  rinsed  in  running  water,  roughly  drained  on  a 
towel,  dried,  and  finally  polished  with  a  selvyt  cloth. 


FIG.  23. — Slides  and  cover-slips,  actual  size. 

If  only  a  few  slides  are  required  for  immediate  use 
a  good  plan  is  to  rub  the  surface  with  jeweler's  emery 
paper  (Hubert's  oo).  A  piece  of  hard  wood  y6X26X 
26  mm.  with  a  piece  of  this  emery  paper  gummed 
tightly  around  it  is  an  exceedingly  useful  article  on 
the  microscope  bench. 

Cover=slips. — The  most  useful  sizes  are  the  19  mm. 
squares  for  ordinary  cover-glass  film  preparations,  and 
38  by  19  mm.  rectangles  for  blood  films  and  serial  sec- 
tions; both  varieties  must  be  of  "No.  i"  thickness, 
which  varies  between  0.15  and  0.22  mm.,  that  they 
may  be  available  for  use  with  the  high-power  immer- 
sion lenses. 

Cover-slips  should  be  cleaned  in  the  following  man- 
ner: 

i .  Drop  the  cover-slips  one  by  one  into  an  enamelled 
iron  pot  or  tall  glass  beaker,  containing  a  10  per  cent, 
solution  of  chromic  acid. 


USED    SLIDES  AND    COVER-SLIPS  23 

2.  Heat  over  a   Bunsen  flame  and  allow  the  acid 
to  boil  gently  for  twenty  minutes. 

NOTE. — A  few  pieces  of  pipe-clay  or  pumice  may  be  placed  in 
the  beaker  to  prevent  the  "spurting"  of  the  chromic  acid. 

3 .  Turn  the  cover-slips  out  into  a  flat  glass  dish  and 
wash  in  running  water  under  the  tap  until  all  trace 
of  yellow  colour  has  disappeared.     During  the  wash- 
ing keep  the  cover-slips  in  motion   by  imparting  a 
rotatory  movement  to  the  dish. 

4.  Wash  in  distilled  water  in  a  similar  manner. 

5.  Wash  in  rectified  spirit. 

6.  Transfer  the  cover-slips,  by  means  of  a  pair  of 
clean  forceps,  previously  heated  in  the  Bunsen  flame 
to  destroy  any  trace  of  grease,  to  a  small  beaker  of 
absolute  alcohol. 

Drain  off  the  alcohol  and  transfer  the  cover-slips, 
by  means  of  the  forceps,  to  a  wide-mouthed  glass  pot, 
containing  absolute  alcohol,  in  which  they  are  to  be 
stored,  and  stopper  tightly. 

NOTE. — After  once  being  placed  in  the  chromic  acid,  the  cover- 
slips  must  on  no  account  be  touched  by  the  fingers. 

Used  Slides  and  Cover=slips. — Used  slides  with  the 
mounted  cover-slip  preparations,  and  cover-slips  used 
for  hanging-drop  mounts,  should,  when  discarded,  be 
thrown  into  a  pot  containing  a  2  per  cent,  solution  of 
lysol. 

After  immersion  therein  for  a  week  or  so,  even  the 
cover-slips  mounted  with  Canada  balsam  can  be  readily 
detached  from  their  slides. 

Slides. — 

1 .  Wash  the  slides  thoroughly  in  running  water. 

2.  Boil  the  slides  in  water  to  which  "sapon"  has 
been  added,  for  half  an  hour. 

3.  Rinse  thoroughly  in  cold  water. 

4.  Dry  and  polish  with  a  dry  cloth. 


24  GLASS  APPARATUS   IN   COMMON    USE 

Cover-slips. — 

1.  Wash  the  cover-slips  thoroughly  in  running  water. 

2.  Boil  the  cover-slips  in  10  per  cent,  solution  of 
chromic  acid,  as  for  new  cover-slips. 

3.  Wash  thoroughly  in  running  water. 

4.  Pick  out  those  cover-slips  which  show  much  ad- 
herent dirty  matter,  and  rub  them  between  thumb 
and  forefinger  under  the  water  tap.     The  dirt  usually 
rubs  off  easily,  as  it  has  become  friable  from  contact 
with  the  chromic  acid. 

5.  Return  all  the  cover-slips  to  the  beaker,  fill  in 
fresh  chromic  acid  solution,  and  treat  as  new  cover- 
slips. 

NOTE. — Test-tubes,  plates,  capsules,  etc.,  which,  from  long  use, 
have  become  scratched  and  hazy,  or  which  cannot  be  cleaned  in 
any  other  way,  may  be  dealt  with  by  immersing  them  in  an 
enamelled  iron  bath,  containing  water  acidulated  to  i  per  cent, 
with  hydrofluoric  acid,  for  ten  minutes,  rinsing  thoroughly  in 
water,  drying,  and  polishing. 

PLUGGING  TEST-TUBES  AND  FLASKS. 

Before  sterilisation  all  test-tubes  and  flasks  must 
be  carefully  plugged  with  cotton-wool,  and  for  this 
purpose  best  absorbent  cotton-wool  (preferably  that 
put  up  in  cylindrical  one-pound  packets  and  inter- 
leaved with  tissue  paper — known  as  surgeons'  wool) 
should  be  employed. 

1.  For  a  test-tube  or  a  small  flask,  tear  a  strip  of 
cotton- wool  some  10  cm.  long  by  2  cm.  wide  from  the 
roll. 

2.  Turn  in  the  ends  neatly  and  roll  the  strip  of  wool 
lightly  between  the  thumb  and  fingers  of  both  hands 
to  form  a  long  cylinder. 

3.  Double  this  at  the  centre  and  introduce  the  now 
rounded  end  into  the  open  mouth  of  the  tube  or  flask. 

4.  Now,   whilst   supporting  the  wool   between  the 
thumb  and  fingers  of  the  right  hand,  rotate  the  test- 


PLUGGING  TEST-TUBES. 


25 


tube  between  those  of  the  left,  and  gradually  screw 
the  plug  of  wool  into  its  mouth  for  a  distance  of  about 
2.5  cm.,  leaving  about  the  same  length  of  wool  pro- 
jecting. 

The  plug  must  be  firm  and  fit  the  tube  or  flask  fairly 
tightly,  sufficiently  tightly  in  fact  to  bear  the  weight 


FIG.  24. — Plugging  test-tubes:  a,  cylinder  of  wool  being  rolled;  &,  cylinder 
of  wool  being  doubled;  c,  cylinder  of  wool  being  inserted  in  tube. 

of  the  glass  plus  the  amount  of  medium  the  vessel  is 
intended  to  contain,  but  not  so  tightly  as  to  prevent 
it  from  being  easily  removed  by  a  screwing  motion 
when  grasped  between  the  fourth,  or  third  and  fourth 
fingers,  and  the  palm  of  the  hand. 

For  a  large  flask  a  similar  but  larger  strip  of  wool 
must  be  taken;  the  method  of  making  and  inserting 
the  plug  is  identical. 


III.  METHODS  OF  STERILISATION. 

STERILISING  AGENTS. 

STERILISATION — i.  e.,  the  removal  or  the  destruction 
of  germ  life — may  be  effected  by  the  use  of  various 
agents.  As  applied  to  the  practical  requirements  of 
the  bacteriological  laboratory,  many  of  these  agents, 
such  as  electricity,  sunlight,  etc.,  are  of  little  value, 
others  are  limited  in  their  applications;  others  again 
are  so  well  suited  to  particular  purposes  that  their  use 
is  almost  entirely  restricted  to  such. 

The  sterilising  agents  in  common  use  are : 
Chemical    Reagents. — Disinfectants    (for  the    disin- 
fection of  glass  and  metal  apparatus  and  of  morbid 
tissues) . 
^  Physical  Agents.     HEAT. — (a)  Dry  Heat: 

1.  Naked   flame    (for  the  sterilisation  of  platinum 
needles,  etc.) . 

2.  Muffle  furnace  (for  the  sterilisation  of  filter  can- 
dles, and  for  the  destruction  of  morbid  tissues) . 

3.  Hot  air  (for  the  sterilisation  of  all  glassware  and 
of  metal  apparatus). 

(b)  Moist  Heat: 

1.  Water  at  56°  C.   (for  the  sterilisation  of  certain 
albuminous  fluids) . 

2.  Water  at  100°  C.  (for  the  sterilisation  of  surgical 
instruments,  rubber  tubing,  and  stoppers,  etc.) . 

3.  Streaming  steam  at  100°  C.  (for  the  sterilisation 
of  media) . 

4.  Superheated  steam  at  115°  C.  or  120°  C.  (for  the 
disinfection  of  contaminated  articles  and  the  destruc- 
tion of  old  cultivations  of  bacteria) . 

26 


CHEMICAL  REAGENTS  27 

FILTRATION. — 

1 .  Cotton-wool  filters  (for  the  sterilisation  of  air  and 
gases) . 

2.  Porcelain  filters   (for  the  sterilisation  of  various 
liquids) . 

METHODS  OF  APPLICATION. 

Chemical  Reagents,  such  as  belong  to  the  class  known 
as  antiseptics  (i.  e.,  substances  which  inhibit  the  growth 
of,  but  do  not  destroy,  bacterial  life),  are  obviously 
useless.  Disinfectants  or  germicides  (i.  e.,  substances 
which  destroy  bacterial  life) ,  on  the  other  hand,  are  of 
value  in  the  disinfection  of  morbid  material,  and  also 
of  various  pieces  of  apparatus,  such  as  pipettes,  pend- 
ing their  cleansing  and  complete  sterilisation  by  other 
processes.  To  this  class  (in  order  of  general  utility) 
belong : 

Lysol,  2  per  cent,  solution; 

Perchloride  of  mercury,  o.i  per  cent,  solution; 

Carbolic  acid,  5  per  cent,  solution ; 

Absolute  alcohol; 

Ether; 

Chloroform ; 

Camphor; 

Thymol; 

Toluol; 

Volatile  oils,  such  as  oil  of  mustard,  oil  of  garlic. 
Formaldehyde  is  a  powerful  germicide,  but  its  pene- 
trating vapor  restricts  its  use.  These  disinfectants  are 
but  little  used  in  the  final  sterilisation  of  apparatus, 
chiefly  on  account  of  the  difficulty  of  effecting  their 
complete  removal,  for  the  presence  of  even  traces  of 
these  chemicals  is  sufficient  to  so  inhibit  or  alter  the 
growth  of  bacteria  as  to  vitiate  subsequent  experi- 
ments conducted  by  the  aid  of  apparatus  sterilised  in 
this  manner. 


28 


METHODS    OF   STERILISATION 


NOTE. — Tubes,  flasks,  filter  flasks,  pipettes,  glass  tubing,  etc., 
may  be  rapidly  sterilised,  in  case  of  emergency,  by  washing,  in 
turn,  with  distilled  water,  perchloride  of  mercury  solution,  alco- 
hol, and  ether,  draining,  and  finally  gently  heating  over  a  gas 
flame  to  completely  drive  off  the  ether  vapor.  Chloroform  or 
other  volatile  disinfectants  may  be  added  to  various  fluids  in 
order  to  effect  the  destruction  of  contained  bacteria,  and  when 
this  has  been  done,  may  be  completely  driven  off  from  the  fluid 
by  the  application  of  gentle  heat. 

Dry  Heat. — The  naked  flame  of  the  Bunsen  burner 
is  invariably  used  for  sterilising  the  platinum  needles 
(which  are'  heated  to  redness)  and  may  be  employed 
for  sterilising  the  points  of  forceps,  or  other  small 

instruments,  cover-glasses,  pi- 
pettes, etc.,  a  very  short  ex- 
posure to  this  heat  being 
sufficient. 

Ether  Flame. — In  an  emer- 
gency small  instruments, 
needles,  etc.,  may  be  sterilised 
by  dipping  them  in  ether  and 
after  removal  lighting  the 
adherent  fluid  and  allowing 
it  to  burn  off  the  surface  of 

FIG.  25. — Muffle  furnace.  . 

the  instruments.  Repeat  the 

process  twice.  It  may  then  be  safely  assumed  that  the 
apparatus  so  treated  is  sterile. 

Muffle  Furnace  (Fig.  2  5)  .—Although  this  form  of 
heat  is  chiefly  used  for  the  destruction  of  the.  dead 
bodies  of  small  infected  animals,  morbid  tissues,  etc., 
it  is  also  employed  for  the  sterilisation  of  porcelain 
filter  candles  (vide  p.  42) . 

Filter  candles  are  disinfected  immediately  after  use 
by  boiling  in  a  beaker  of  water  for  some  fifteen  or 
twenty  minutes.  This  treatment,  however,  leaves  the 
dead  bodies  of  the  bacteria  upon  the  surface  and  block- 
ing the  interstices  of  the  filter. 

To  destroy  the  organic  matter  and  prepare  the  filter 
candle  for  further  use  proceed  as  follows : 


DRY   HEAT 


29 


1.  Roll  each  bougie  up  in  a  piece  of  asbestos  cloth, 
secure  the  ends  of  the  cloth  with  a  few  turns  of  cop- 
per wire,  and  place  inside  the  muffle  (a  small  muffle 
76X88X163   mm.  will  hold  perhaps  four  small  filter 
candles) . 

2.  Light  the  gas  and  raise  the  contents  of  the  muffle 
to  a  white  heat;  maintain  this  temperature  for  five 
minutes. 

3.  Extinguish  the  gas,   and  when  the  muffle  has 
become  quite  cold  remove  the  filter  candles,  and  store 
them    (without  removing  the  asbestos  wrappings)  in 
sterile  metal  boxes.     . 

NOTE. — The  too  rapid  cooling  of  the  candles,  such  as  takes  place 
if  they  are  removed  from  the  muffle  before  it  has  cooled  down  to 
the  room  temperature,  may  give  rise  to  microscopic  cracks  and 
flaws  which  will  effectually  destroy  their  efficiency. 

Hot  Air. — Hot  air  at  150°  C.  destroys  all  bacteria, 
spores,  etc.,  in  about  thirty  minutes;  a  momentary 
exposure  to  a  temperature  of  175°  to  180°  C.  will 
effect  the  same  result  and  offers  the  more  convenient 
method  of  sterilisation.  This  method  is  only  appli- 
cable to  glass  and  metallic  substances,  and  the  small 
bulk  of  cotton-wool  comprised  in  the  test-tube  plugs, 
etc.  Large  masses  of  fabric  are  not  effectually  steril- 
ised by  dry  heat — short  of  charring — as  its  power  of 
penetration  is  not  great. 

Sterilisation  by  hot  air  is  effected  in  the  hot-air  oven 
(Fig.  1 8).  This  is  a  rectangular,  double- walled  metal 
box,  mounted  on  a  stand  and  heated  from  below  by  a 
large  Bunsen  burner.  The  interior  of  the  oven  is 
provided  with  loose  shelves  upon  which  the  articles 
to  be  sterilised  are  arranged,  either  singly  or  packed 
in  square  wire  baskets  or  crates,  kept  specially  for  this 
purpose.  One  of  the  sides  is  hinged  to  form  a  door. 
The  central  portion  of  the  metal  bottom,  on  which 
the  Bunsen  flame  would  play,  is  cut  away,  and  replaced 
by  firebrick  plates,  which  slide  in  metal  grooves  and 


METHODS    OF    STERILISATION 


are  easily  replaced  when  broken  or  worn  out.  The 
top  of  the  oven  is  provided  with  a  perforated  ventilator 
slide  and  two  tubulures,  the  one  for  the  reception  of  a 
centigrade  thermometer  graduated  to  200°  or  250°  C., 
the  other  for  a  thermo-regulator.  An  ordinary  mer- 
curial thermo-regulator  may  be  used  but  it  is  prefer- 
able to  employ  a  regulating  capsule  of  the  Hearson 
type  (see  p.  219)  with  a  spring  arm  adjusted  to  the  lever 
so  that  when  the  boiling-point  of  the  capsule  (e.g., 


FIG.  26. — Hot-air  oven. 

175°  C.)  is  reached  the  gas  supply  is  absolutely  cut  off 
and  the  jet  cannot  again  be  lighted  until  the  spring 
arm  has  been  readjusted  by  hand.  The  thermo- 
regulator  is  by  no  means  a  necessity,  and  may  be 
replaced  by  a  large  bore  thermometer  with  a  sliding 
platinum  point,  connected  with  an  electric  bell,  which 
can  be  easily  adjusted  to  ring  at  any  given  temperature. 
Even  if  the  steriliser  is  provided  with  the  capsule 
regulator  above  described  the  contact  thermometer 
should  also  be  fitted. 


DRY   HEAT  3! 

To  USE  THE  HOT-AIR  OVEN. — 

1.  Place  the  crates  of  test-tubes,  metal  cases  con- 
taining plates  and  pipettes,  loose  apparatus,  etc.,  inside 
the   oven,    taking   particular   care  that   none   of    the 
cotton- wool  plugs  are  in  contact  with  the  walls,  other- 
wise the  heat  transmitted  by  the  metal  will  char  or 
even  flame  them. 

To  prepare  a  wire  crate  for  the  reception  of  test-tubes,  etc., 
cover  the  bottom  with  a  layer  of  thick  asbestos  cloth;  or  take 
some  asbestos  fibre,  moisten  it  with  a  little  water  and  knead  it 
into  a  paste;  plaster  the  paste  over  the  bottom  of  the  crate, 
working  it  into  the  meshes  and  smoothing  the  surface  by  means  of 
a  pestle.  When  several  crates  have  been  thus  treated,  place  them 
inside  the  hot-air  oven,  close  the  door,  open  the  ventilating  slide, 
light  the  gas,  and  run  the  temperature  of  the  interior  up  to  about 
1 60°  C.  After  an  interval  of  ten  minutes  extinguish  the  gas,  open 
the  oven  door,  and  allow  the  contents  to  cool.  The  asbestos  now 
forms  a  smooth,  dry,  spongy  layer  over  the  bottom,  which  will 
last  many  months  before  needing  renewal,  and  will  considerably 
diminish  the  loss  of  tubes  from  breakage. 

Copper  cylinders  and  large  test-tubes  intended  for  the  reception 
of  pipettes  are  prepared  in  a  similar  manner,  in  order  to  protect 
the  points  of  these  articles  from  injury. 

2.  Close  the  oven  door,   and  open  the  ventilating 
slide,  in  order  that  any  moisture  left  in  the  tubes,  etc., 
may  escape;  light  the  gas  below;  set  the  electric  alarm 
to  ring  at  1 00°  C. 

3.  When  the  temperature  of  the  oven  has  reached 
100°  C.,  close  the  ventilating  slide ;  reset  the  alarm  to 
ring  at  175°  C. 

4.  Run  the  temperature  up  to  1 75°  C. 

5.  Extinguish  the  gas  at  once,  and  allow  the  appa- 
ratus to  cool. 

6.  When  the  temperature  of  the  interior,  as  recorded 
by  the  thermometer,   has  fallen  to   60°   C. — but  not 
before — the  door  may  be  opened  and  the  sterile  articles 
removed  and  stored  away. 

NOTE. — Neglect  of  this  precautionary  cooling  of  the  oven  to 
60°  C.  will  result  in  numerous  cracked  and  broken  tubes. 


32  METHODS    OF   STERILISATION 

On  removal  from  the  oven,  the  cotton-wool  plugs 
will  probably  be  slightly  brown  in  colour. 

Metal  instruments,  such  as  knives,  scissors,  and 
forceps,  may  be  sterilised  in  the  hot-air  oven  as  de- 
scribed above,  but  exposure  to  175°  C.  is  likely  to 
seriously  affect  the  temper  of  the  steel  and  certainly 
blunts  the  cutting  edges.  If,  however,  it  is  desired 
to"  sterilise  surgical  instruments  by  hot  air,  they  should 
be  packed  in  a  metal  box,  or  boxes,  and  heated  to 
130°  C.  and  retained  at  that  temperature  for  about 
thirty  minutes. 

Moist  Heat. — Water  at  56°  C. — This  temperature,  if 
maintained  for  thirty  minutes,  is  sufficient  to  destroy 
the  vegetative  forms  of  bacteria,  but  has  practically 
no  effect  on  spores.  Its  use  is  limited  to  the  sterilisa- 
tion of  such  albuminous  " fluid"  media  as  would  coagu- 
late at  a  higher  temperature. 

METHOD. — 

1.  Fit  up  a  water-bath,  heated  .by  a  Bunsen  flame 
which  is  controlled  by  a  thermo-regulator,  so  that  the 
temperature  of  the  water  remains  at  56°  C. 

2.  Immerse  the  tubes  or  flasks  containing  the  albu- 
minous fluid  in  the  water-bath  so  that  the  upper  level 
of  such  fluid  is  at  least  2  cm.  below  the  level  of  the 
water.     (The  temperature  of  the  bath  will  now  fall 
somewhat,  but  after  a  few  minutes  will  again  rise  to 
56°  C). 

3.  After  thirty  minutes'   exposure  to    56°   C.,   ex- 
tinguish the  gas,  remove  the  tubes  or  flasks  from  the 
bath,  and  subject  them  to  the  action  of  running  water 
so  that  their  contents  are  rapidly  cooled. 

4.  The  vegetative  forms  of  bacteria  present  in  the 
liquid  being  killed,  stand  it  for  twenty-four  hours  in 
a  cool,  dark  place ;  at  the  end  of  that  time  some  at  least 
of  such  spores  as  may  be  present  will  have  germinated 
and  assumed  the  vegetative  form. 


MOIST    HEAT 


33 


5.  Destroy  these  new  vegetative  forms  by  a  similar 
exposure  to  56°  C.  on  the  second  day,  whilst  others, 
of  slower  germination,  may  be  caught  on  the  third  day, 
and  so  on. 

6.  In  order  to  ensure  thorough  sterilisation,  repeat 
the  process  on  each  of  six  successive  days. 

This  method  of  exposing  liquids  to  a  temperature 
of  56°  C.  in  a  water-bath  for  half  an  hour  on  each  of 
six  successive  days  is  termed  fractional  sterilisation. 

Water  at  100°  C.  destroys  the  vegetative  forms  of 
bacteria  almost  instantaneously,  and  spores  in  from 


FIG,  27. — Water  sterilizer. 

five  to  fifteen  minutes.  This  method  of  sterilisation 
is  applicable  to  the  metal  instruments,  such  as  knives, 
forceps,  etc.,  used  in  animal  experiments;  syringes, 
rubber  corks,  rubber  and  glass  tubing,  and  other  small 
apparatus,  and  is  effected  in  what  is  usually  spoken  of 
as  the  "  water  steriliser"  (Fig  27). 

This  is  a  rectangular  copper  box,  26  cm.  long,  18  cm. 
wide,  and  12  cm.  deep,  mounted  on  legs,  heated  from 
below  by  a  Bunsen  or  radial  gas  burner,  and  containing 
a  movable  copper  wire  tray,  2  cm.  smaller  in  every 

3 


34 


METHODS    OF   STERILISATION 


dimension  than  the  steriliser  itself,  and  provided  with 
handles.     The  top  of  the  steriliser  is  hinged  to  form  a  lid. 

METHOD.— 

1.  Place  the  instruments,   etc.,   to  be  sterilised  in- 
side the  copper  basket,  and  replace  the  basket  in  the 
steriliser. 

2.  Pour  a  sufficient  quantity  of  water  into  the  ster- 
iliser, shut  down  the  lid,  and  light  the  gas  below. 


FIG.  28. — Koch's  steriliser. 


FIG.  29. — Arnold's  steriliser. 


3.  After  the  water  has  boiled  and  steam  has  been 
issuing  from  beneath  the  lid  for  at  least  ten  minutes, 
extinguish  the  gas,  open  the  lid,  and  lift  out  the  wire 
basket  by  its  handles  and  rest  it  diagonally  on  the 
walls  of  the  steriliser;  the  contained  instruments,  etc., 
are  now  sterile  and  ready  for  use. 

4.  After  use,   or  when  accidentally  contaminated, 
replace  the  instruments  in  the  basket  and  return  that 
to  the  steriliser;  completely  disinfect  by  a  further  boil- 
ing for  fifteen  minutes. 

5.  After  disinfection,  and  whilst  still  hot,  take  out 


MOIST    HEAT  35 

the  instruments,  dry  carefully  and  at  once,  and  return 
them  to  their  store  cases. 

Streaming  steam — i.  e.,  steam  at  100°  C. — destroys 
the  vegetative  forms  of  bacteria  in  from  fifteen  to 
twenty  minutes,  and  the  sporing  forms  in  from  one 
to  two  hours.  This  method  is  chiefly  used  for  the 
sterilisation  of  the  various  nutrient  media  intended  for 
the  cultivation  of  bacteria,  and  is  carried  out  in  a 
steam  kettle  of  special  construction,  known  as  Koch's 
steam  steriliser  (Fig.  28)  or  in  one  of  its  many  modifi- 
cations, the  most  efficient  of  which  is  Arnold's  (Fig.  29) . 

The  steam  steriliser  in  its  simplest  form  consists  of 
a  tall  tinned-iron  or  copper  cylindrical  vessel,  divided 
into  two  unequal  parts  by  a  movable  perforated  metal 
diaphragm,  the  lower,  smaller  portion  serving  for  a 
water  reservoir,  and  the  upper  part  for  the  reception  of 
wire  baskets  containing  the  articles  to  be  sterilised. 
The  vessel  is  closed  by  a  loose  conical  lid,  provided 
with  handles,  and  perforated  at  its  apex  by  a  tubu- 
lure;  it  is  mounted  on  a  tripod  stand  and  heated 
from  below  by  a  Bunsen  burner.  The  more  elaborate 
steriliser  is  cased  with  felt  or  asbestos  board,  and  pro- 
vided with  a  water  gauge,  also  a  tap  for  emptying  the 
water  compartment. 

To  USE  THE  STEAM  STERILISER.— 

1.  Fill  the  water  compartment  to  the  level  of  the 
perforated  diaphragm,  place  the  lid  in  position,  and 
light  the  Bunsen  burner. 

2.  After  the  water  has  boiled,  allow  sufficient  time 
to  elapse  for  steam  to  replace  the  air  in  the  sterilising 
compartment,   as  shown  by  the  steam  issuing  in  a 
steady,  continuous  stream  from  the  tubulure  in  the  lid. 

3.  Remove  the  lid,  quickly  lower  the  wire  basket 
containing  media  tubes,  etc.,  into  the  sterilising  com- 
partment until  it  rests  on  the  diaphragm,  and  replace 
the  lid. 


36  METHODS    OF    STERILISATION 

4.  After  an  interval  of  twenty  minutes  in  the  case 
of  fluid  media,  or  thirty  minutes  in  the  case  of  solid 
media,  take  off  the  lid  and  remove  the  basket  with  its 
contents. 

5.  Now,  but  not  before,  extinguish  the  gas. 

NOTE. — After  removing  tubes,  flasks,  etc.,  from  the  steam 
steriliser,  they  should  be  at  once  separated  freely  in  order  to  pre- 
vent moisture  condensing  upon  the  cotton-wool  plugs  and  soaking 
through  into  the  interior  of  the  tubes. 

This  treatment  will  destroy  any  vegetative  forms  of 
bacteria ;  during  the  hours  of  cooling  any  spores  present 
will  germinate,  and  the  young  organisms  will  be  de- 
stroyed by  repeating  the  process  twenty-four  hours 
later;  a  third  sterilisation  after  a  similar  interval 
makes  assurance  doubly  sure. 

The  method  of  sterilising  by  exposure  to  streaming 
steam  at  100°  C.  for  twenty  minutes  on  each  of  three 
consecutive  days  is  termed  discontinuous  or  inter- 
mittent sterilisation. 

Exposure  to  steam  at  100°  C.  for  a  period  of  one  or 
two  hours,  or  continuous  sterilisation,  cannot  always  be 
depended  upon  and  is  therefore  not  to  be  recommended. 

Superheated  steam — i.  e.,  steam  under  pressure  (see 
Pressure-temperature  table,  Appendix,  page  500)  in 
sealed  vessels  at  a  temperature  of  115°  C. — will  destroy 
both  the  vegetative  and  the  sporing  forms  of  bacteria 
within  fifteen  minutes ;  if  the  pressure  is  increased,  and 
the  temperature  raised  to  120°  C.,  the  same  end  is  at- 
tained in  ten  minutes.  This  method  was  formerly  em- 
ployed for  the  sterilisation  of  media  (and  indeed  is  so 
used  in  some  laboratories  still),  but  most  workers 
now  realise  that  media  subjected  to  this  high  tem- 
perature undergo  hydrolytic  changes  which  render 
them  unsuitable  for  the  cultivation  of  the  more  deli- 
cate micro-organisms.  The  use  of  superheated  steam 
should  be  restricted  almost  entirely  to  the  disinfection 
of  such  contaminated  articles,  old  cultivations,  etc., 


MOIST    HEAT 


37 


as  cannot  be  dealt  with  by  dry  heat  or  the  actual 
furnace.  Sterilisation  by  means  of  superheated  steam 
is  carried  out  in  a  special  boiler — Chamberland's 
autoclave  (Fig.  30).  The  autoclave  consists  of  a  stout 
copper  ylinder,  provided  with  a  copper  or  gun-metal 
lid,  which  is  secured  in  place  by  means  of  bolts  and 
thumbscrews,  the  joint  between  the  cylinder  and  its 
lid  being  hermetically  sealed  by  the  interposition  of  a 
rubber  washer.  The  cover  is  perforated  for  a  branched 
tube  carrying  a  vent  cock,  a  manometer,  and  a  safety 
valve.  The  copper  boiler  is  mounted  in  the  upper  half 
of  a  cylindrical  sheet-iron  case — two  concentric  cir- 
cular rows  of  Bunsen  burners,  each  circle  having  an 
independent  gas-supply,  occupying  the  lower  half. 
In  the  interior  of  the  boiler  is  a  large  movable  wire 
basket,  mounted  on  legs,  for  the  reception  of  the 
articles  to  be  sterilised. 

To  USE  THE  AUTOCLAVE. — 

1.  Pack   the   articles   to   be   sterilised  in   the   wire 
basket. 

2.  Run  water  into  the  boiler  to  the  level  of  the  bot- 
tom of  the  basket;  also  fill  the  contained  flasks  and 
tubes  with  water. 

3.  See  that  the  rubber  washer  is  in  position,  then 
replace  the  cover  and  fasten  it  tightly  on  to  the  auto- 
clave by  means  of  the  thumbscrews. 

4.  Open  the  vent  cock  and  light  both  rings  of  burners. 

5.  When  steam  is  issuing  in  a  steady,  continuous 
stream  from  the  vent  tube,  shut  off  the  vent  cock  and 
extinguish  the  outer  ring  of  gas  burners. 

6.  Wait  until  the  index  of  the  manometer  records  a 
temperature  of  120°  C.,  then  regulate  the  gas  and  the 
spring  safety  valve  in  such  a  manner  that  this  tem- 
perature  is   just   maintained,    and   leave  it   thus   for 
twenty  minutes.     In  the  more  expensive  patterns  of 
autoclave  this  regulation  of  the  safety  valve  is  carried 


38  METHODS    OF    STERILISATION 

out  automatically,  the  manometer  being  fitted  with  an 
adjustable  pointer  which  can  be  set  to  any  required 
pressure-temperature  and  so  arranged  that  when  the 
index  of  the  manometer  coincides  with  the  adjustable 
hand  the  safety  valve  is  opened. 

7.  Extinguish   the  gas   and   allow   the   manometer 
index  to  fall  to  zero. 


FIG.  30. — Chamberland's  Autoclave. 

8.  Now  open  the  vent  cock  slowly,  and  allow  the 
internal    pressure    to    adjust    itself    to    that    of    the 
atmosphere. 

9.  Remove  the  cover  and  take  out  the  sterilised 
contents. 

Sterilisation  Periods. — An  exceedingly  useful  device 
for  the  timing  of  sterilisation  periods  (and  indeed  for 
many  other  operations  in  the  laboratory)  is  the 

ELECTRIC  SIGNAL  TIMING  CLOCK. 

This  is  a  clock  of  American  type  in  which  the  face 
is  surrounded  by  a  metal  plate  having  a  series  of  60 


TIMING  STERILISATION  PERIODS  39 

holes  at  equal  distances  apart,  corresponding  to  the 
minutes  on  the  dial.  This  plate  is  connected  with 
one  of  the  poles  of  a  dry  battery,  the  other  pole  of 
which  is  connected  to  the  metal  case  of  the  clock  for 
the  purpose  of  actuating  an  ordinary  magnet  alarm 
bell.  In  the  centre  of  each  of  the  holes  in  the  plate 
a  metal  rod  is  fixed,  which  then  passes  through  an 


FIG.  31. — Electric  signal  timing  clock. 

insulating  ring  and  projects  inside  the  clock  face, 
where  it  makes  contact  with  the  hour  hand.  The 
clock  is  mounted  on  a  heavy  base,  with  a  key-board 
containing  20  numbered  plugs.  If  one  of  the  plugs  is 
inserted  in  a  hole  in  the  plate  it  makes  contact  with 
the  rod,  and  when  the  hour  hand  of  the  clock  touches 
the  other  end  the  circuit  is  completed  and  the  bell 
starts  ringing.  The  period  of  this  friction  contact  is 
approximately  20  seconds.  The  clock  can  therefore 
be  used  for  electrically  noting  the  periods  of  time 
from  one  minute  by  multiples  of  one  minute  up  to  one 
hour. 


4O  METHODS    OF   STERILISATION 

Filtration. — (a)  Cotton-wool  Filter. — Practically  the 
only  method  in  use  in  the  laboratory  for  the  sterilisa- 
tion of  air  or  of  a  gas  is  by  filtration  through  dry  cotton- 
wool or  glass-wool,  the  fibres  of  which  entangle  the 
micro-organisms  and  prevent  their  passage. 

Perhaps  the  best  example  of  such  a  filter  is  the  cotton- 
wool plug  which  closes  the  mouth  of  a  culture  tube. 
Not  only  does  ordinary  diffusion  take  place  through  it, 
but  if  a  tube  plugged  in  the  usual  manner  with  cotton- 
wool is  removed  from  the  hot  incubator,  the  tempera- 
ture of  the  contained  air  rapidly  falls  to  that  of  the 


FIG.  32. — Cotton-wool  air  filter. 

laboratory,  and  a  partial  vacuum  is  formed;  air  passes 
into  the  tube,  through  the  cotton-wool  plug,  to  restore 
the  equilibrium,  and,  so  long  as  the  plug  remains  dry, 
in  a  germ-free  condition.  If,  however,  the  plug  be- 
comes moist,  either  by  absorption  from  the  atmos- 
phere, or  from  liquids  coming  into  contact  with  it, 
micro-organisms  (especially  the  mould  fungi)  com- 
mence to  multiply,  and  the  long  thread  forms  rapidly 
penetrate  the  substance  of  the  plug,  and  gain  access  to 
and  contaminate  the  interior  of  the  tube. 

METHOD. — 

If  it  is  desired  to  sterilise  gases  before  admission  to 
a  vessel  containing  a  pure  cultivation  of  a  micro- 
organism, as,  for  instance,  when  forcing  a  current  of 
oxygen  over  or  through  a  broth  cultivation  of  the 
diphtheria  bacillus,  this  can  be  readily  effected  as 
follows : 


FILTRATION  41 

'i.  Take  a  length  of  glass  tubing  of,  say,  1.5  cm. 
diameter,  in  the  centre  of  which  a  bulb  has  been  blown, 
fill  the  bulb  with  dry  cotton-wool  (Fig.  32),  wrap  a 
layer  of  cotton-wool  around  each  end  of  the  tube,  and 
secure  in  position  with  a  turn  of  thin  copper  wire  or 
string;  then  sterilise  the  piece  of  apparatus  in  the  hot- 
air  oven. 

2.  Prepare  the  cultivation  in  a  Ruffer  or  Woodhead 
flask  (Fig.  33)  the  inlet  tube  of  which  has  its  free 
extremity  enveloped  in  a  layer  of  cotton- wool,  secured 


FIG.  33.— Ruffer's  flask. 

by  thread  or  wire,  whilst  the  exit  tube  is  plugged  in 
the  usual  manner. 

3.  Sterilise  a  short  length  of  rubber  tubing  by  boil- 
ing.    Transfer  it  from  the  boiling  water  to  a  beaker 
of  absolute  alcohol. 

4.  When  all  is  ready  remove  the  rubber  tube  from 
the    alcohol    by  means  of  a  pair    of    forceps,   drain 
it  thoroughly,  and  pass  through  the  flame  of  a  Bunsen 
burner  to  burn  off  the  last  traces  of  alcohol. 

5.  Remove  the  cotton-wool  wraps  from  the  entry 
tube  of  the  flask  and  from  one  end  of  the  filter  tube 
and  rapidly  couple  them  up  by  means  of  the  sterile 
rubber  tubing. 


42  METHODS    OF   STERILISATION 

6.  Connect  the  other  end  of  the  bulb  tube  with  the 
delivery  tube  from  the  gas  reservoir. 

The  gas  in  its  passage  through  the  dry  sterile  cotton- 
wool in  the  bulb  of  the  filter  tube  will  be  freed  from 
any  contained  micro-organisms  and  will  enter  the 
flask  in  a  sterile  condition. 

(b)  Porcelain  Filter. — The  sterilisation  of  liquids  by 
filtration  is  effected  by  passing  them  through  a  cylin- 
drical vessel,  closed  at  one  end  like  a  test-tube,  and 
made  either  of  porous  "biscuit"  porcelain,  hard-burnt 
and  unglazed  (Chamberland  system),  or  of  Kieselguhr, 
a  fine  diatomaceous  earth  (Berkefeld  system),  and 
termed  a  "  bougie  "  or  "  candle  "  (Fig.  34) . 

NOTE. — In  selecting  candles  for  use  in  the  laboratory  avoid  those 
with  metal  fittings,  since  during  sterilisation  cracks  develop  at  the 
junction  of  the  metal  and  the  siliceous  material  owing  to  the  un- 
equal expansion. 

In  this  method  the  bacteria  are  retained  in  the  pores 
of  the  filter  while  the  liquid  passes  through  in  a  germ- 
free  condition. 

It  is  obvious  that  to  be  effective  the  pores  of  the 
filter  must  be  extremely  minute,  and  therefore  the  rate 
of  filtration  will  usually  be  slow.  Chamberland  filter 
candles  possess  finer  channels  than  Berkefeld  candles 
and  consequently  filter  much  more  slowly.  To  over- 
come this  disadvantage,  either  aspiration  or  pressure, 
or  a  combination  of  these  two  forces,  may  be  employed 
to  hasten  the  process. 

Doultons  white  porcelain  filters  it  may  be  noted  are 
as  efficient  as  the  Chamberland  candles  and  filter 
rather  more  rapidly. 

Apparatus  Required. — 

1.  Separatory  funnel  containing  the  unfiltered  fluid. 

2.  Sterile    filter  candle    (Fig.  34),  the  open  end  fitted  with  a 
rubber  stopper  (Fig.  34,  a)  perforated  to  receive  the  delivery  tube 
of  the  separatory  funnel,  and  its  neck  passed  through  a  large  rubber 
washer  (Fig.  34,  6)  which  fits  the  mouth  of  the  filter  flask. 

3.  Sterile  filter  flask  of  suitable  size,  for  the  reception  of  the 
filtered  fluid,  its  mouth  closed  by  a  cotton- wool  plug. 


FILTRATION 


43 


4.  Water  injector  Sprengel  (see  Fig.  38,  c)   pump,  or  Geryk's 
pump  (an  air  pump  on  the  hydraulic  principle,  sealed  by  means 
of  low  vapor-tension  oil,  Fig.  35). 

If  this  latter  is  employed,  a  Wulff's  bottle,  fitted  as  a  wash- 
bottle  and  containing  sulphuric  acid,  must  be  in- 
terposed between  the  filter  flask  and  the  pump,  in 
order  to  prevent  moist  air  reaching  the  oil  in  the 
pump. 

5.  Air  filter  (vide  page  40)  sterilised. 

6.  Pressure  tubing. 

7.  Screw  clamps  (Fig.  36). 

METHOD. — 


i. 


Couple  the  exhaust  pipe  of  the  suc- 


tion  pump  with  the  lateral  tube  of  the  FIG.  34.— Force- 
filter  flask  (first  removing  the  cotton-  lainfiltercandle- 
wool  plug  from  this  latter),  by  means  of  pressure 
tubing,  interposing,  if  necessary,  the  wash-bottle  of 
sulphuric  acid. 


FIG.  35. — Geryk  air  pump. 


44 


METHODS    OF    STERILISATION 


2.  Remove  the  cotton-wool  plug  from  the  neck  of 
the  filter  flask  and  adjust  the  porcelain  candle  in  its 
place. 


FIG.  36. — Screw  clamps. 

3.  Attach  the  nozzle  of  the  separatory  funnel  to  the 
filter  candle  by  means  of  the  perforated  rubber  stopper 
(Fig.  37)- 


FIG.  37. — Apparatus  arranged  for  filtering — aspiration. 

4.  Open  the  tap  of  the  funnel,  and  exhaust  the  air 
from  the  filter  flask  and  wash-bottle;  maintain  the 
vacuum  until  the  nitration  is  complete. 

5.  When  the  filtration  is  completed  close  the  tap  of 


FILTRATION  45 

the  funnel ;  adjust  a  screw  clamp  to  the  pressure  tubing 
attached  to  the  lateral  branch  of  the  filter  flask;  screw 
it  up  tightly,  and  disconnect  the  acid  wash-bottle. 

6.  Attach  the  air  filter  to  the  open  end  of  the  pressure 
tubing;  open  the  screw  clamp  gradually,  and  allow 
filtered  air  to  enter  the  flask,  to  abolish  the  negative 
pressure. 

7.  Detach  the  rubber  tubing  from  the  lateral  branch 
of  the  flask,  flame  the  end  of  the  branch  in  the  Bunsen, 
and  plug  its  orifice  with  sterile  cotton- wool. 

8.  Remove  the  filter  candle  from  the  mouth  of  the 
flask,  flame  the  mouth,  and  plug  the  neck  with  sterile 
cotton- wool. 

9.  Disinfect  the  filter  candle  and  separatory  funnel 
by  boiling. 

If  it  is  found  necessary  to  employ  pressure  in  addi- 
tion to  or  in  place  of  suction,  insert  a  perforated  rubber 
stopper  into  the  mouth  of  the  separatory  funnel  and 
secure  in  position  with  copper  wire;  next  fit  a  piece  of 
glass  tubing  through  the  stopper,  and  connect  the 
external  orifice  with  an  air-pressure  pump  of  some 
kind  (an  ordinary  foot  pump  such  as  is  employed  for 
inflating  bicycle  tyres  is  one  of  the  most  generally  use- 
ful, for  this  purpose)  or  with  a  cylinder  of  compressed 
air  or  other  gas. 

In  order  to  filter  a  large  bulk. of  fluid  very  rapidly  it 
is  necessary  to  use  a  higher  pressure  than  glass  would 
stand,  and  in  these  cases  the  metal  receptacle  designed 
by  Pakes  (Fig.  38,  a),  to  hold  the  filter  candle  itself  as 
well  as  the  fluid  to  be  filtered,  should  be  employed. 
(A  vacuum  must  also  be  maintained  in  the  filter  flask, 
by  means  of  an  exhaust  pump,  during  the  entire 
process.) 

This  piece  of  apparatus  consists  of  a  brass  cylinder, 
capacity  2500  c.c.,  with  two  shoulders;  and  an  opening 
in  the  neck  at  each  end,  provided  with  screw  threads. 

A  nut  carrying  a  pressure  gauge  fits  into  the  top 


40  METHODS    OF   STERILISATION 

screw;  and  into  the  bottom  is  fitted  a  brass  cylinder 
carrying  the  filter  candle  and  prolonged  downwards 
into  a  delivery  tube.  Leakage  is  prevented  by  means 
of  rubber  washers. 

Into  the  top  shoulder  a  tube  is  inserted,  bent  at 
right  angles  and  provided  with  a  tap.  All  the  brass- 
work  is  tinned  inside  (Fig.  38,  a).  In  use  the  reservoir 
is  generally  mounted  on  a  tripod  stand. 

To  Sterilise.— 

i.  Insert  the  filter  candle  into  its  cylinder  and  screw 
this  loosely  on. 


FIG.  38. — Fakes'  filtering  reservoir — pressure  and  aspiration. 

2.  Wrap  a  layer  of  cotton- wool  around  the  delivery 
tube  and  fasten  in  position. 

3.  Remove  the  nut  carrying  the  pressure  gauge  and 
plug  the  neck  with  cotton- wool. 


FILTRATION  47 

4.  Heat  the  whole  apparatus  in  the  autoclave  at 
120°  C.  for  twenty  minutes. 

METHOD.— 

1.  Remove  the  apparatus  from  the  autoclave,  and 
allow  it  to 'cool. 

2.  Screw  home  the  box  carrying  the  bougie. 

3 .  Set  the  apparatus  up  in  position,  with  its  delivery 
tube  (from  which  the  cotton-wool  wrapping  has  been 
removed)  passing  through  a  perforated  rubber  stopper 
in  the  neck  of  a  filter  flask. 


FIG.  39. — Closed  candle  arranged  for  filtering. 

4.  Fill  the  fluid  to  be  filtered  into  the  cylinder  and 
screw  on  the  nut  carrying  the  pressure  gauge.     (This 
nut  should  be  immersed  in  boiling  water  for  a  few 
minutes  previous  to  screwing  on,  in  order  to  sterilise  it.) 

5.  Connect  the  horizontal  arm  of  the  entry  tube 
with  a  cylinder  of  compressed  oxygen  (or  carbon  di- 
oxide, Fig.  38,  6),  by  means  of  pressure  tubing. 

6.  Connect  the  lateral  arm  of  the  filter  flask  with 
the  exhaust   pump   (Fig.  38,  c)  and  start  the  latter 
working. 


48  METHODS    OF    STERILISATION 

7.  Open  the  tap  of  the  gas  cylinder;  then  open  the 
tap  on  the  entry  tube  of  the  filter  cylinder  and  raise 
the  pressure  in  its  interior  until  the  desired  point 
is  recorded  on  the  manometer.  Maintain  this  pressure, 
usually  one  or  one  and  a  half  atmospheres,  until  filtra- 
tion is  completed,  by  regulating  the  tap  on  the  entry 
tube. 

Some  forms  of  filter  candle  are  made  with  the  open 
end  contracted  into  a  delivery  nozzle,  which  is  glazed. 
In  this  case  the  apparatus  is  fitted  up  in  a  slightly 
different  manner;  the  fluid  to  be  filtered  is  contained 
in  an  open  cylinder  into  which  the  candle  is  plunged, 
while  its  delivery  nozzle  is  connected  with  the  filter 
flask  by  means  of  a  piece  of  flexible  pressure  tubing 
(previously  sterilised  by  boiling),  as  in  figure  39. 


IV.  THE  MICROSCOPE. 

THE  essentials  of  a  microscope  for  bacteriological 
work  may  be  briefly  summed  up  as  follows : 

The  instrument,  of  the  monocular  type,  must  be  of 


FIG.  40. — Microscope  stand. 

good  workmanship  and  well  finished,  rigid,  firm,  and 
free  from  vibration,  not  only  when  upright,  but  also 
when  inclined  to  an  angle  or  in  the  horizontal  position. 
The  various  joints  and  movements  must  work  smoothly 
and  precisely,  equally  free  from  the  defects  of  "loss 
of  time"  and  "slipping."  All  screws,  etc.,  should  con- 
4  49 


50  THE    MICROSCOPE 

form  to  the  Royal  Microscopical  Society's  standard. 
It  must  also  be  provided  with  good  lenses  and  a  suffi- 
ciently large  stage.  The  details  of  its  component  parts, 
to  which  attention  must  be  specially  directed,  are  as 
follows : 

1.  The  Base  or  Foot  (Fig.  40,  a). — Two  elementary 
forms — the  tripod  (Fig.  41,  a)  and  the  vertical  column 


FIG.  41. — Foot,  three  types. 

set  into  a  plate  known  as  the  "horse-shoe"  (Fig 
41,  b) — serve  as  the  patterns  for  countless  modifica- 
tions in  shape  and  size  of  this  portion  of  the  stand. 
The  chief  desiderata — stability  and  ease  of  manipula- 
tion— are  attained  in  the  first  by  means  of  the  "  spread  " 
of  the  three  feet,  which  are  usually  shod  with  cork;  in 
the  second,  by  the  dead  weight  of  the  foot-plate.  The 
tripod  is  mechanically  the  more  correct  form,  and  for 
practical  use  is  much  to  be  preferred.  Its  chief  rival, 
the  Jackson  foot  (Fig.  41,  £),  is  based  upon  the  same 
principle,  and  on  the  score  of  appearance  has  much  to 
recommend  it. 

2.  The  body  tube  (Fig.  40,  b)  may  be  either  that 
known  as  the  "long"  or  "English"  (length  250  mm.), 
or  the  "short"  or  "Continental"  (length  160  mm.). 
Neither  length  appears  to  possess  any  material  advan- 
tage over  the  other,  but  it  is  absolutely  necessary  to 
secure  objectives  which  have  been  manufactured  for 
the  particular  tube  length  chosen.  In  the  high-class 
microscope  of  the  present  day  the  body  tube  is  usually 


THE    FINE   ADJUSTMENT  51 

shorter  than  the  Continental,  but  is  provided  with  a 
draw  tube  which,  when  fully  extended,  gives  a  tube 
length  greater  than  the  English,  thus  permitting  the 
use  of  either  form  of  objective. 

For  practical  purposes  the  tube  length  =  distance  from  the 
end  of  the  nosepiece  to  the  eyeglass  of  the  ocular.  This  is  the 
measurement  referred  to  in  speaking  of  "long"  or  "short"  tube. 


FIG.  42. — Coarse  adjustment. 


FIG.  43. — Fine  adjustment. 


3.  The  coarse  adjustment  (Fig.  40,  c)  should  be  a 
rack-and-pinion  movement,  steadiness  and  smoothness 
of  action  being  secured  by  means  of  accurately  fitting 
dovetailed  bearings  and  perfect  correspondence  between 
the  teeth  of  the  rack  and  the  leaves  of  the  pinion  (Fig. 
42).     Also  provision  should  be  made  for  taking  up  the 
"slack"  (as  by  the  screws  A  A,  Fig.  42). 

4.  The  fine  adjustment  (Fig.   40,  d)   should  on  no 
account  depend  upon  the  direct  action  of  springs,  but 


52  THE    MICROSCOPE 

should  be  of  the  lever  pattern,  preferably  the  Nelson 
(Fig.  43).  In  this  form  the  unequal  length  of  the 
arms  of  the  lever  secures  very  delicate  movement,  and, 
moreover,  only  a  small  portion  of  the  weight  of  the 
body  tube  is  transmitted  to  the  thread  of  the  vertical 
screw  actuating  the  movement. 

A  spindle  milled  head  (Fig.  44)  will  be  found  a  very 
useful  device  to  have  fitted  in  place  of  the  ordinary 
milled  head  controlling  the  fine  adjustment.  In  this 
contrivance  the  axis  of  the  milled  head  is  prolonged 
upward  in  a  short  column,  the  diameter  of  which  is 
one-sixth  of  that  of  the  head.  The  spindle 
can  be  rapidly  rotated  between  the  fingers 
for  medium  power  adjustments  while  the 
larger  milled  head  can  be  slowly  moved 
when  focussing  high  powers. 

5.  The  stage  (Fig.  40,  e)  should  be  square 
in  shape  and  large  in  area — at  least  12  cm. 
—flat  and  rigid,  in  order  to  afford  a  safe 
support  for  the  Petri  dish  used  for  plate 
Spindle  head  to  cultivations;  and  should  be  supplied,  with 
fine  adjust-  spring  clips  (removable  at  will)  to  secure 

the  3  by  i  glass  slides. 

A  mechanical  stage  must  be  classed  as  a  necessity 
rather  than  a  luxury  so  far  as  the  bacteriologist  is 
concerned,  as  when  working  with  high  powers,  and 
especially  when  examining  hanging-drop  specimens, 
it  is  almost  impossible  to  execute  sufficiently  delicate 
movements  with  the  fingers.  In  selecting  a  mechan- 
ical stage,  preference  should  be  given  to  one  which 
forms  an  integral  part  of  the  instrument  (Fig.  45) 
rather  than  one  which  needs  to  be  clamped  on  to  an 
ordinary  plain  stage  every  time  it  is  required,  and  its 
traversing  movements  should  be  controlled  by  sta- 
tionary milled  heads  (Fig.  45,  A  A'}.  The  shape  of  the 
aperture  is  a  not  unimportant  point;  it  should  be 
square  to  allow  of  free  movement  over  the  substage 


DIAPHRAGM. 


53 


condenser.  The  mechanical  stage  should  be  tapped 
for  three  (removable)  screw  studs  to  be  used  in  place 
of  the  sliding  bar,  so  that  if  desired  the  Vernier  finder 


FIG.  45. — Mechanical  stage. 

(Fig.  45,  BBf),  such  as  is  usually  fitted  to  this  class  of 
stage,  or  a  Malt  wood  finder,  may  be  employed. 

6.  Diaphragm. — Separate    single    diaphragms   must 


FIG.  46. — Iris  diaphragm. 

be  avoided;  a  revolving  plate  pierced  with  different 
sized  apertures  and  secured  below  the  stage  is  prefer- 
able, but  undoubtedly  the  best  form  is  the  "iris" 


54  THE    MICROSCOPE 

diaphragm  (Fig.  46)   which  enters  into  the  construc- 
tion of  the  substage  condenser. 

7.  The  substage  condenser  is  a  necessary  part  of  the 
optical  outfit.  Its  purpose  is  to  collect  the  beam  of 
parallel  rays  of  light  reflected  by  the  plane  mirror,  by 
virtue  of  a  short  focus  system  of  lenses,  into  a  cone  of 
large  aperture  (reducible  at  will  by  means  of  an  iris 
diaphragm  mounted  as  a  part  of  the  condenser), 
which  can  be  accurately  focussed  on  the  plane  of  the 
object.  This  focussing  must  be  performed  anew  for 
each  object,  on  account  of  the  variation  in  the  thick- 
ness of  the  slides. 

The  form  in  most  general  use  is  that  known  as  the 
Abbe  (Fig.  47)  and  consists  of  a  plano-convex  lens 
mounted  above  a  biconvex  lens.  This  combination 
is  carried  in  a  screw-centering  holder  known  as  the 
substage  below  the  stage  of  the  microscope  (Fig.  40,  /) , 

and  must  be  accurately  adjusted 
so  that  its  optical  axis  coincides 
with  that  of  the  objective.     Ver- 
k     tical    movement    of    the    entire 
pi  substage  apparatus  effected  by 
means  of  a  rack  and  pinion  is  a 
FiG.47.-Op^partofAbbe  decided    advantage,    and    some 

means  should   be  provided  for 

temporarily  removing  the  condenser  from  the  optical 
axis  of  the  microscope. 

With  the  oil  immersion  objective,  however,  an 
achromatic  condenser,  giving  an  illuminating  cone  of 
about  0.9,  should  be  used  if  the  full  value  of  the  lens 
is  to  be  obtained.  It  is  generally  assumed  that  a  good 
objective  requires  an  illuminating  cone  equivalent  to 
two-thirds  of  its  numerical  aperture.  The  best  Abbe 
condenser  transmits  a  cone  of  about  .45  whilst  the 
aperture  of  the  TV  inch  immersion  lenses  of  different 
makers  varies  from  i.o  to  1.4,  hence,  the  efficiency  of 
these  lenses  is  much  curtailed  if  the  condenser  is  merely 


OCULARS  AND    OBJECTIVES  55 

the  Abbe.  These  improved  condensers  must  be 
absolutely  centered  to  the  objective  and  capable  of 
very  accurate  focussing  otherwise  much  of  their  value 
is  lost. 

8.  Mirrors. — Below  the  substage   condenser  is   at- 
tached a  gymbal  carrying  a  reversible  circular  frame 
with  a  plane  mirror  on  one  side  and  a  concave  mirror 
on  the  other  (Fig.  40,  g).     The  plane  mirror  is  that 
usually  employed,    but  occasionally,   as  for  example 
when  using  low  powers  and  with  the  condenser  racked 
down  and  thrown  out  of  the  optical  axis,  the  concave 
mirror  is  used. 

9.  Oculars,    or    Eyepieces. — Those    known    as    the 
Huyghenian  oculars  (Fig.  48)  will  be  sufficient  for  all 
ordinary  work  without  resorting  to  the  more  expen- 
sive   "compensation"    oculars.     Two    or  three,  mag- 
nifying the  "real"  image  (formed  by  the  objective) 
four,  six,  or  eight  times  respectively,  form  a  useful 
equipment. 

As  an  accessory  Ehrlich's  Eyepiece  is  a  very  useful 
piece  of  apparatus  when  the  enumeration  of  cells  or 
bacteria  has  to  be  carried  out.  This  is  an  ordinary 
eyepiece  fitted  with  an  adjustable  square  diaphragm 
operated  by  a  lever  projecting  from  the  side  of  the 
mount.  Three  notches  are  made  in  one  of  the  sides  of 
the  square  and  by  moving  the  lever  the  square  aperture 
can  be  reduced  to  three-quarters,  one-half  or  one-quar- 
ter of  the  original  size. 

10.  Objectives. — Three  objectives  are  necessary:  one 
for  low-power  work — e.g.,   i  inch,  f  inch,  or  \  inch; 
one  for  high-power  work — e.  g.,  -£%  inch  oil  immer- 
sion lens;  and  an  intermediate  "medium-power"  lens — 
e.  g.,  J  inch  or  J  inch  (dry).     These  lenses  must  be 
carefully  selected,  especial  attention  being  paid  to  the 
following  points: 

(a)  Correction  of  Spherical  Aberration. — Spherical 
aberration  gives  rise  to  an  ill-defined  image,  due  to  the 


THE   MICROSCOPE 


central    and    peripheral    rays    focussing    at    different 
points. 

(b)  Correction  of  Chromatic  Aberration. — Chromatic 
aberration  gives  rise  to  a  coloured  fringe  around  the 
edges  of  objects  due  to  the  fact  that  the  different- 
coloured  rays  of  the  spectrum  possess  varying  refran- 
gibilities  and  that  a  simple  lens  acts  toward  them  as  a 
prism. 

(c)  Flatness  of  Field. — The  ideal  visual  field  would  be 
large  and,  above  all,  flat;  in  other  words,  objects  at  the 

periphery  of  the  field  would  be  as 
distinctly  "  in  focus  "  as  those  in  the 
centre.  Unfortunately,  however, 
this  is  an  optical  impossibility  and 
the  field  is  always  spherical  in  shape. 
Some  makers  succeed  in  giving  a 
larger  central  area  that  is  in  focus 
at  one  time  than  others,  and  al- 
though this  may  theoretically  cause 
an  infinitesimal  sacrifice  of  other 
qualities,  it  should  always  be 
sought  for.  Successive  zones  and 
the  entire  peripheral  ring  should 
come  into  focus  with  the  alteration 
of  the  fine  adjustment.  This  simultaneous  sharpness 
of  the  entire  circle  is  an  indication  of  the  perfect  cen- 
tering of  the  whole  of  the  lenses  in  the  objective. 

(d)  Good  Definition. — Actual  magnification  is,  within 
limits,  of  course,  of  less  value  than  clear  definition  and 
high  resolving  power,  for  it  is  upon  these  properties 
we  depend  for  our  knowledge  of  the  detailed  structure 
of  the  objects  examined. 

(e)  Numerical    Aperture    (N.    A.). — The    numerical 
aperture  may  be  defined,  in  general  terms,  as  the  ratio 
of  the  effective  diameter  of  the  back  lens  of  the  objective 
to  its  equivalent  focal  length.     The  determination  of 
this  point  is  a  process  requiring  considerable  technical 


FIG.  48. — Huyghenian 
eyepiece. 


ACCESSORIES  57 

skill  and  mathematical  ability,  and  is  completely 
beyond  the  powers  of  the  average  microscopist.1 

Although  with  the  increase  in  power  it  is  correspond- 
ingly difficult  to  combine  all  these  corrections  in  one 
objective,  they  are  brought  to  a  high  pitch  of  excel- 
lence in  the  present-day  " achromatic"  objectives,  and 
so  remove  the  necessity  for  the  use  of  the  higher  priced 
and  less  durable  apochromatic  lenses. 

In  selecting  objectives  the  best  "test"  objects  to 
employ  are: 

1 .  A  thin  (one  cell  layer) ,  even 

1  'blood  film,  "stained with Jenner's    for 
or  Romanowsky's  stain. 

2.  A  thin  cover -slip  prepara- 
tion  of   a   young   cultivation   of 

B.  diphtheria  (showing  segment  a-  \  for 


2  n     iff 

¥  >  V 


r,  r 

-rJ'  Oil 


\" 
"8 


dry 
''oil 


tion)     stained    with    methylene- 
blue. 

Accessories. — Eye  Shade  (Fig.  49). — This  piece  of 
apparatus  consists  of  a  pear-shaped  piece  of  blackened 
metal  or  ebonite,  hinged  to  a  collar  which  rotates  on 
the  upper  part  of  the  body  tube  of  the  microscope. 
It  can  be  used  to  shut  out  the  image  of  surrounding 
objects  from  the  unoccupied  eye,  and  when  carrying 
out  prolonged  observations  will  be  found  of  real  service. 

Nosepiece. — Perhaps  the  most  useful  accessory  is  a 
nosepiece  to  carry  two  of  the  objectives  (Fig.  50),  or, 
better  still,  all  three  (Fig.  51).  This  nosepiece,  pref- 
erably constructed  of  aluminium,  must  be  of  the 
covered-in  type,  consisting  of  a  curved  plate  attached 
to  the  lower  end  of  the  body  tube — a  circular  aperture 
being  cut  to  correspond  to  the  lumen  of  that  tube.  To 

1  Its  importance  will  be  realised,  however,  when  it  is  stated  in  the  words  of 
the  late  Professor  Abbe:  "The  numerical  aperture  of  a  lens  determines  all 
its  essential  qualities;  the  brightness  of  the  image  increases  with  a  given 
magnification  and  other  things  being  equal,  as  the  square  of  the  aperture;  the 
resolving  and  denning  powers  are  directly  related  to  it,  the  focal  depth  of 
differentiation  of  depths  varies  inversely  as  the  aperture,  and  so  forth." 


THE    MICROSCOPE 


the  under  surface  of  this  plate  is  pivoted  a  similarly 
curved  plate,  fitted  with  three  tubulures,  each  of  which 
carries  an  objective.  By  rotating  the  lower  plate  each 
of  the  objectives  can  be  brought  successively  in  to  the 
optical  axis  of  the  microscope. 


FIG.  49. — Eye  shade. 

For  critical  work  and  particularly  for  photo-microg 
raphy,  however,  the  interchangeable  nosepiece  is  by 
no  means  perfect  as  it  is  next  to  impossible  to  secure 
accurate  centreing  of  each  lens  in  the  optical  axis. 
For  special  purposes,  therefore,  it  is  necessary  to  employ 


FIG.  50. — Double  nosepiece. 


FIG.  51  — Triple  nosepiece 


a  special  nosepiece  such  as  that  made  by  Zeiss  or  Leitz 
into  which  each  objective  slides  on  its  own  carrier  and 
upon  which  it  is  accurately  centred. 

Warm  Stage  (Pig.  52). — This  is  a  flat  metal  case 
containing  a  system  of  tubes  through  the  interior  of 
which  water  of  any  required  temperature  can  be  cir- 


WARM   STAGE  59 

culated.  It  is  made  to  clamp  on  to  the  stage  of  the 
microscope  by  the  screws  A  A',  and  is  perforated  with 
a  large  hole  coinciding  with  the  optical  axis  of  the 
microscope;  a  short  tube  B,  projecting  from  one  end 
of  the  warm  stage  permits  water  of  the  desired  temper- 
ature to  be  conducted  from  a  reservoir  through  a  length 
of  rubber  tubing  to  the  interior  of  the  stage  and  a 
similar  tube  at  the  other  end  B'  of  the  stage  allows 
exit  to  the  waste  water.  By  raising  the  temperature 
of  hanging-drop  preparations,  etc.,  placed  upon  it, 
above  that  of  the  surrounding  atmosphere,  the  warm 
stage  renders  possible  exact  observations  on  spore 
germination,  hanging-drop  cultivations,  etc. 


FIG.  52. — Warm  stage. 

A  better  form  is  the  electrical  hot  stage  designed  by 
Lorrain  Smith;1  it  requires  the  addition  of  a  lamp 
resistance  and  sliding  rheostat,  also  a  delicate  ammeter 
reading  to  .01  of  an  ampere.  It  consists  of  a  wooden 
frame  supporting  a  flat  glass  bulb  with  a  long  neck  bent 
upward  at  an  obtuse  angle  (Fig.  53).  The  bulb  is 
filled  with  liquid  paraffin,  which  rises  in  the  open  neck 
when  expanded  by  heat.  The  neck  also  accommo- 
dates the  thermometer.  Two  coils  of  manganin  wire 
run  in  the  paraffin  at  opposite  sides  of  the  bulb  (out- 
side the  field  of  vision),  coupled  to  brass  terminals  on 
the  wooden  frame  by  platinum  wire  fused  into  the 
glass.  The  resistance  of  the  two  coils  in  series  is 

1  Made  by  Mr.  Otto  Baumbach,  10,  Lime  Grove,  Manchester. 


60  THE   MICROSCOPE 

about  10  ohms.  A  current  of  2\  amperes  is  needed, 
and  is  conducted  to  the  coils  in  the  stage  through  the 
rheostat.  With  the  help  of  the  ammeter  any  desired 
temperature  can  be  obtained  and  maintained,  up  to 
about  200°  C.  If  immersion  oil  contact  is  made 
between  the  top  lens  of  the  condenser  and  the  lower 
surface  of  the  bulb,  this  stage  works  very  well  indeed 
with  the  ---inch  oil  immersion  lens. 


FIG.  53. — Lorrain  Smith's  warm  stage. 

Dark  Ground  or  Paraboloid  Condenser. — This  is  an 
immersion  substage  condenser  of  high  aperture  by 
means  of  which  unstained  objects  such  as  bacteria 
can  be  shown  as  bright  white  particles  upon  a 
dense  black  background.  The  central  rays  of  light 
are  blocked  out  by  means  of  an  opaque  stop  while  the 
peripheral  rays  are  reflected  from  the  paraboloidal  sides 
of  the  condenser  and  refracted  by  the  object  viewed. 
To  obtain  the  best  results  with  this  type  of  condenser 
a  powerful  illuminant — such  as  a  small  arc  lamp  or  an 
incandescent  gas  lamp — is  needed,  together  with 
picked  slides  of  a  certain  thickness  (specified  for  the 


MICROMETRY  6 1 

particular  make  of  condenser  but  generally  i  mm.  and 
specially  thin  cover-glasses  (not  more  than  0.17  mm. 
The  objective  must  not  have  a  higher  NA  than  i.o, 
consequently  immersion  lenses  must  be  fitted  with  an 
internal  stop  to  cut  down  the  aperture. 

Micrometer. — Some  form  of  micrometer  for  the  pur- 
pose of  measuring  bacteria  and  other  objects  is  also 
essential.  Details  of  those  in  general  use  will  be 
found  in  the  following  pages. 

Object  Marker  (Fig.  54). — This  is  an  exceedingly 
useful  piece  of  apparatus.  Made  in  the  form  of  an 
objective,  the  lenses  are  replaced  by  a  diamond  point, 
set  slightly  out  of  the  centre,  which 
can  be  rotated  by  means  of  a  milled 
plate.  Screwed  on  to  the  nosepiece 
in  place  of  the  objective,  rotation  of 
the  diamond  point  will  rule  a  small 
circle  on  the  object  slide  to  perma- 
nently record  the  position  of  an  in- 
teresting portion  of  the  specimen. 
The  diamond  is  mounted  on  a  spring  j 
which  regulates  the  pressure,  and  1 

the  size  of  the  circle  can  be  adjusted  FlG-  54--Diamond  Ob- 
ject marker, 
by  means  of  a  lateral  screw. 

METHODS  OF  MICROMETRY. 

The  unit  of  length  as  applied  to  the  measurement 
of  microscopical  objects  is  the  one-thousandth  part  of 
a  millimetre  (o.ooi  mm.),  denominated  a  micron  (some- 
times, and  erroneously,  referred  to  as  a  micro-milli- 
metre), and  indicated  in  writing  by  the  Greek  letter 
fjL.  Of  the  many  methods  in  use  for  the  measurement 
of  bacteria,  three  only  will  be  here  described,  viz. : 

(a)    By  means  of  the  Camera  Lucida. 

(6)    By  means  of  the  ocular  or  Eyepiece  Micrometer. 

(c)  By  means  of  the  Filar  Micrometer  (Ramsden's 
micrometer  eyepiece) . 


62  THE   MICROSCOPE 


each  of  these  methods  a  stage  micrometer  is 
necessary.  This  is  a  3  by  i  inch  glass  slip  having  en- 
graved on  it  a  scale  divided  to  hundredths  of  a  milli- 
metre (o.oi  mm.),  every  tenth  line  being  made  longer 
than  the  intervening  ones,  to  facilitate  counting;  and 
from  these  engraved  lines  the  measurement  in  every 
case  is  evaluated.  A  cover-glass  is  cemented  over 
the  scale  to  protect  it  from  injury. 


FIG.  55. — Camera  lucida,  Abbe  pattern. 

(a)    By  means  of  the  Camera  Lucida. 

1.  Attach  a  camera  lucida  (of  the  Wollaston,  Beale, 
or  Abbe   pattern)    (Fig.   55)   to   the   eyepiece  of   the 
microscope. 

2.  Adjust  the  micrometer  on  the  stage  of  the  micro- 
scope and  accurately  focus  the  divisions. 

3.  Project  the  scale  of  the  stage  micrometer  on  to  a 
piece  of  paper  and  with  pen  or  pencil  sketch  in  the 
magnified  image,  each  division  of  which  corresponds 
to  10  fi.     Mark  on  the  paper  the  optical  combination 
(ocular  objective  and  tube  length)  employed  to  pro- 
duce this  particular  magnification. 


THE  EYEPIECE  MICROMETER  63 

4.  Repeat  this  procedure  for  each  of  the  possible 
combinations  of  oculars  and  objectives  fitted  to  the 
microscope  supplied,  and  carefully  preserve  the  scales 
thus  obtained. 

To  measure  an  object  by  this  method  simply  project 
the  image  on  to  the  scale  corresponding  to  the  par- 
ticular optical  combination  in  use  at  the  moment. 
Read  off  the  number  of  divisions  it  occupies  and  ex- 
press them  as  micra. 

In  place  of  preserving  a  scale  for  each  optical  com- 
bination, the  object  to  be  measured  and  the  microm- 
eter scale  may  be  projected  and  sketched,  in  turn,  on 
the  same  piece  of  paper,  taking  particular  care  that 
the  centre  of  the  eyepiece  is  25  cm.  from  the  paper  on 
which  the  divisions  are  drawn. 


FIG.  56. — Eyepiece  micro- 
meter, ordinary. 


FIG.  57. — Eyepiece  micrometer,  net. 


(b)   By  means  of  the  Eyepiece  Micrometer. 

The  eyepiece  micrometer  is  a  circular  glass  disc 
having  engraved  on  it  a  scale  divided  to  tenths  of  a 
millimetre  (o.i  mm.)  (Fig.  56),  or  the  entire  surface 
ruled  in  o.i  mm.  squares  (the  net  micrometer)  (Fig.  57). 
It  can  be  fitted  inside  the  mount  of  any  ocular  just 
above  the  aperture  of  the  diaphragm  and  must  be 
adjusted  exactly  in  the  focus  of  the  eye  lens. 

Some  makers  mount  the  glass  disc  together  with  a 
circular  cover-glass  in  such  a  way  that  when  placed  in 
position  in  any  Huyghenian  eyepiece  of  their  own 
manufacture,  the  scale  is  exactly  in  focus  for  normal 


64  THE   MICROSCOPE 

vision.  Special  eyepieces  are  also  obtainable  having 
a  sledging  adjustment  to  the  eye  lens  for  focussing 
the  micrometer. 

The  value  of  one  division  of  the  micrometer  scale 
must  first  be  ascertained  for  each  optical  combination 
by  the  aid  of  the  stage  micrometer,  thus : 

1.  Insert  the  eyepiece  micrometer  inside  the  ocular 
and  adjust  the  stage  micrometer  on  the  stage  of  the 
microscope. 

2 .  Focus  the  scale  of  the  stage  micrometer  accurately ; 
the  lines  will  appear  to  be  immediately  below  those  of 
the  eyepiece  micrometer.     Make  the  lines  on  the  two 
micrometers  parallel  by  rotating  the  ocular. 

3.  Make  two  of  the  lines  on  the  ocular  micrometer 
coincide  with  those  bounding  one  division  of  the  stage 
micrometer;  this  is  effected  by  increasing  or  diminish- 
ing the  tube  length;  and  note  the  number  of  included 
divisions. 

4.  Calculate  the  value  of  each  division  of  the  eye- 
piece micrometer  in  terms  of  /*,  by  means  of  the  fol- 
lowing formula : 

x=io  y. 

Where  x  =  the  number  of  included  divisions  of  the 

eyepiece  micrometer. 

y  =  the  number  of  included  divisions  of  the 
stage  micrometer. 

5.  Note  the  optical  combination  employed  in  this 
experiment  and  record  it  with  the  calculated  microm- 
eter value. 

Repeat  this  process  for  each  of  the  other  combina- 
tions. Carefully  record  the  results. 

To  measure  an  object  by  this  method  read  off  the 
number  of  divisions  of  the  eyepiece  micrometer  it 
occupies  and  express  the  result  in  micra  by  a  refer- 
ence to  the  standard  value  for  the  particular  optical 
combination  employed. 


THE    EYEPIECE    MICROMETER  65 

Zeiss  prepares  a  compensating  eyepiece  micrometer 
for  use  with  his  apochromatic  objectives,  the  divisions 
of  which  are  so  computed  that  (with  a  tube  length  of 
1 60  mm.)  the  value  of  each  is  equivalent  to  as  many 
micro,  as  there  are  millimetres  in  the  focal  length  of  the 
objective  employed. 

Wright's  Eikonometer  is  really  a  modification  of 
the  eyepiece  micrometer  for  rapidly  measuring  micro- 
scopical objects  by  direct  inspection,  having  previously 
determined  the  magnifying  power  of  the  particular 
optical  combination  employed.  It  is  a  small  piece  of 
apparatus  resembling  an  eyepiece,  with  a  sliding  eye 
lens,  which  can  be  accurately  focussed  on  a  micrometer 
scale  fixed  within  the  instrument.  When  placed  over 
the  microscope  ocular  the  divisions  of  this  scale  measure 
the  actual  size  of  the  virtual  image  in  millimetres. 

In  order  to  use  this  instrument  for  direct  measure- 
ment, it  is  first  necessary  to  determine  the  magnifying 
power  of  each  combination  of  ocular,  tube  length  and 
objective. 

Place  a  stage  micrometer  divided  into  hundredths 
of  a  millimetre  on  the  microscope  stage  and  focus 
accurately. 

Rest  the  eikonometer  on  the  eyepiece.  Observa- 
tion through  the  eikonometer  shows  its  micrometer 
scale  superposed  on  the  image  of  the  stage  micrometer. 

Rotate  the  eikonometer  until  the  lines  on  the  two 
scales  are  parallel,  and  make  the  various  adjustments 
to  ensure  that  two  lines  on  the  eikonometer  scale 
coincide  with  two  lines  on  the  stage  micrometer. 

For  the  sake  of  illustration  it  may  be  assumed  that 
five  of  the  divisions  on  the  stage  micrometer  accu- 
rately fill  one  of  the  divisions  of  the  eikonometer  scale ; 
this  indicates  a  magnifying  power  of  500  as  the  con- 
stant for  that  particular  optical  combination,  and  a 
record  should  be  made  of  the  fact. 

The  magnification  constants  of  the  various  other 
5 


66 


THE   MICROSCOPE 


optical  combinations  should  be  similarly  made  and 
recorded. 

To  measure  any  object   subsequently  it  should  be 
first  focussed  carefully  in  the  ordinary  way. 

The  eikonometer  should  then  be  applied  to  the  eye- 
piece and  the  size  of  the  object  read  off  on  the  eiko- 
nometer scale  as  millimetres,  and  the  actual  size  calcu- 
lated by  dividing  the  observed  size  by  the  magnification 
constant  for  the  particular  optical  combination  em- 
ployed in  the  observation. 

(c)   By  means  of  the  filar  micrometer. 

The  Filar  or  cobweb  Micrometer  (Ramsden's  microm- 


FIG.  58. — Ramsden's  Filar  micrometer. 


FIG.  59. — Ramsden's 
micrometer  field,  a,  fixed 
wire;  6,  reference  wire 
(fixed) ;  c,  travelling  wire. 


eter  eyepiece  (Fig.  58)  consists  of  an  ocular  having  a 
fine  "fixed"  wire  stretching  horizontally  across  the 
field  (Fig.  59),  a  vertical  reference  wire — fixed — ad- 
justed at  right  angles  to  the  first ;  and  a  fine  wire,  paral- 
lel to  the  reference  wire,  which  can  be  moved  across 
the  field  by  the  action  of  a  micrometer  screw;  the 
drum  head  is  divided  into  one  hundred  parts,  which 
successively  pass  a  fixed  index  as  the  head  is  turned. 
In  the  lower  part  of  the  field  is  a  comb  with  the  inter- 
vals between  its. teeth  corresponding  to  one  complete 
revolution  of  this  screw-head. 


ILLUMINANTS  67 

As  in  the  previous  method,  the  value  of  each  division 
of  the  micrometer  scale  (i.  e.t  the  comb)  must  first  be 
determined  for  each  optical  combination.  This  is 
effected  as  follows: 

1.  Place  the  filar  micrometer  and  the  stage  microm- 
eter in  their  respective  positions. 

2.  Rotate  the  screw  of  the  filar  micrometer  until  the 
movable  wire  coincides  with  the  fixed  one,  and  the 
index  marks  zero  on  the  drum  head.     (If  when  the 
drum  head  is  at  zero  the  two  wires  do  not  exactly  coin- 
cide they  must  be  adjusted  by  loosening  the  drum  screw 
and  resetting  the  drum.) 

3.  Focus  the  scale  of  each  micrometer  accurately, 
and  make  the  lines  on  them  parallel. 

4.  Rotate  the  head  of  the  micrometer  screw  until 
the  movable  line  has  transversed  one  division  of  the 
stage  micrometer.     Note  the  number  of  complete  revo- 
lutions (by  means  of  the  recording  comb)  and  the  frac- 
tions of  a  revolution  (by  means  of  scale  on  the  head 
of  the  micrometer  screw) ,  which  are  required  to  meas- 
ure the  o.o  i  mm. 

5.  Make  several  such  estimations  and  average  the 
results. 

6.  Note  the  optical  combination  employed  in  this 
experiment  and  record  it  carefully,  together  with  the 
micrometer  value  in  terms  of  /*. 

7.  Repeat   this   process   for   each   of   the   different 
optical  combinations  and  record  the  results. 

To  measure  an  object  by  this  method,  simply  note 
the  number  of  revolutions  and  fractions  of  a  revolu- 
tion of  the  screw-head  required  to  traverse  such  object 
from  edge  to  edge,  and  express  the  result  as  micro, 
by  reference  to  the  recorded  values  for  that  particular 
optical  combination. 

Microscope  Illuminant. — In  tropical  and  subtropical 
regions  diffuse  daylight  is  the  best  illuminant.  In 
temperate  climes  however  daylight  of  the  desirable 


68  THE    MICROSCOPE 

quantity  is  not  always  available,  and  recourse  must  be 
had  to  oil  lamps,  gas  lamps — preferably  those  with 
incandescent  mantles — and  electricity;  and  of  these 
the  last  is  undoubtedly  the  best.  A  handy  lamp 
holder  which  can  be  manufactured  in  the  laboratory 
is  shown  in  Fig.  60.  It  consists  of  a  base  board 
weighted  with  lead  to  which  is  attached  the  ordinary 
domestic  lamp  holder,  and  behind  this  is  fastened  a 


FIG.  60. — Electric  microscope  lamp. 

curved  sheet-iron  reflector.  An  obscured  metal  fila- 
ment lamp  of  about  16  candle  power  gives  the  most 
suitable  light,  and  if  monochromatic  light  is  needed, 
the  blue  grease  pencil  is  streaked  over  the  side  of  the 
lamp  nearest  the  microscope;  the  current  is  switched 
on  and  when  the  glass  bulb  is  warm,  rubbing  with  a  wad 
of  cotton-wool  will  readily  distribute  the  blue  greasy 
material  in  an  even  film  over  the  ground  glass. 


V.  MICROSCOPICAL  EXAMINATION  OF  BAC- 
TERIA AND  OTHER  MICRO-FUNGI. 

APPARATUS  AND  REAGENTS  USED  IN  ORDINARY 
MICROSCOPICAL  EXAMINATION. 

The  following  comprises  the  essential  apparatus 
and  reagents  for  routine  work  with  which  each  student 
should  be  provided. 

1.  India-rubber  " change-mat"  upon  which  cover- 
glasses  may  be  rested  during  the  process  of  staining. 

2.  Squares  of  blotting  paper  about  10  cm.,  for  dry- 
ing cover-slips  and  slides. 

(The  filter  paper  known  as  "  German  lined " — a 
highly  absorbent,  closely  woven  paper,  having  an  even 


FIG.  61. — Disinfectant  Jar. 

surface  and  no  loose  "fluff"  to  adhere  to  the  specimens 
— is  the  most  useful  for  this  purpose.) 

3.  Glass  jar  filled  with  2  per  cent,  lysol  solution  for 
the  reception  of  infected  cover-glasses  and  infected 
pipettes,  etc. 

69 


MICROSCOPICAL   EXAMINATION   OF   BACTERIA 


4.  A  square  glazed  earthenware  box  with  a  loose 
lining    containing    2    per   cent,    lysol  solution  for  the 
reception  of  infected  material  and  used  slides.     The 
bottom  of  the  lining  is  perforated  so  that  when  full  the 
lining  and  its  contents  can  be  lifted  bodily  out  of  the 
box,  when  the  disinfectant  solution  drains  away  and  the 
slides,  etc.,  can  easily  be  emptied  out.     The  empty  lin- 
ing is  then  returned  to  the  box  with  its  disinfectant 
solution  (Fig.  61). 

5.  Bunsen    burner    provided    with     "peep-flame" 
by-pass. 

6.  Porcelain  trough  holding  five  or  six  hanging-drop 
slides  (Fig.  62). 


FIG.  62. — Hanging-drop  slides:  a,  Double  cell  seen  from  above;  b    single 
cell  seen  from  the  side. 


The  best  form  of  hanging-drop  slide  is  a  modification 
of  Boettcher's  glass  ring  slide,  and  is  prepared  by 
cementing  a  circular  cell  of  tin,  13  to  15  mm.  diameter, 
and  i  to  2  mm.  in  height,  to  the  centre  of  a  3  by  i  slip 
by  means  of  Canada  balsam.  It  is  often  extremely 
convenient  to  have  two  of  these  cells  cemented  close 
together  on  one  slide  (Fig.  62,  a). 

Another  form  of  hanging-drop  slide  is  made  in  which  a  circular 
or  oval  concavity  or  "cell"  is  ground  out  of  the  centre  of  a  3  by  i 
slip.  These  are  more  expensive,  less  convenient  to  work  with, 
and  are  more  easily  contaminated  by  drops  of  material  under 
examination,  and  should  be  carefully  avoided. 


MICROSCOPICAL  EXAMINATION   OF  BACTERIA  71 

7.  Three  aluminium  rods  (Fig.  63),  each  about  25 
cm.  long  and  carrying  a  piece  of  0.015  gauge  platino- 
iridium  wire  7.5  cm.  in  length.  The  end  of  one  of  the 
wires  is  bent  round  to  form  an  oval  loop,  of  about  i 
mm.  in  its  short  diameter,  and  is  termed  a  loop  or  an 
oese;  the  terminal  3  or  4  mm.  of  another  wire  is  flat- 
tened out  by  hammering  it  on  a  smooth  iron  surface 
to  form  a  " spatula";  the  third  is  left  untouched  or  is 
pointed  by  the  aid  of  a  file.  These  instruments  are 


FIG.  63. — Ends  of  platinum  rods,     a,  loop;  b,  spatula;  c,  needle. 

used  for  inoculating  culture  tubes  and  preparing  speci- 
mens for  microscopical  examination. 

The  method  of  mounting  these  wires  may  be  de- 
scribed as  follows: 

Take  a  piece  of  aluminium  wire  25  cm.  long  and 
about  0.25  cm.  in  diameter,  and  drill  a  fine  hole  com- 
pletely through  the  wire  about  a  centimetre  from  one 
end.  Sink  a  straight  narrow  channel  along  one  side 
of  the  wire,  in  its  long  axis,  from  the  hole  to  the  nearest 
end,  shallow  at  first,  but  gradually  becoming  deeper. 

On  the  opposite  side  of  the  wire  make  a  short  cut, 
2  mm.  in  length,  leading  from  the  hole  in  the  same 
direction.  [The  use  of  a  fine  dental  drill  and  small 
circular  saw,  worked  by  a  dental  motor  facilitates  the 
manufacture  of  these  aluminium  handled  instruments.] 

Now  pass  one  end  of  the  platinum  wire  through  the 
hole,  turn  up  about  2  mm.  at  right  angles  and  press 


72  MICROSCOPICAL   EXAMINATION    OF   BACTERIA 

the  short  piece  into  the  short  cut.  Turn  the  long  end 
of  the  wire  sharply,  also  at  right  angles,  and  sink  it 
into  the  long  channel  so  that  it  emerges  from  about 
the  centre  of  the  cut  end  of  the  aluminium  wire  (Fig. 
63).  A  few  sharp  taps  with  a  watch  maker's  hammer 
will  now  close  in  the  sides  of  the  two  channels  over  the 
wire  and  hold  it  securely. 


FIG.  64. — Platinum  rod  in  aluminium  handle — method  of  mounting. 

The  platinum  wire  may  be  fused  into  the  end  of  a  piece  of  glass 
rod,  but  such  a  handle  is  vastly  inferior  to  aluminium  and  is  not 
to  be  recommended. 

8.  Two   pairs   of  sharp-pointed   spring  forceps    (10 
cm.  long),  one  of  which  must  be  kept  perfectly  clean 
and  reserved  for  handling  clean  cover-slips,  the  other 
being  for  use  during  staining  operations. 

9.  A  box  of  clean  3  by  i  glass  slips. 

10.  A  glass  capsule  with  tightly  fitting  (ground  on) 
glass    lid,    containing     clean    cover-slips    in    absolute 
alcohol. 

11.  One  of  Faber's  "grease  pencils"  (yellow,  red,  or 
blue)  for  writing  on  glass. 

12.  A  wooden  rack  (Fig.  65)  with  twelve  drop-bottles 
(Fig.  66)  each  60  c.c.  capacity,  containing 

Aniline  water. 

Gentian  violet,  saturated  alcoholic  solution. 

Lugol's  (Gram's)  iodine. 

Absolute  alcohol. 

Methylene-blue,  ] 

Fuchsin,  basic     )  saturated  alcoholic  solution. 


MICROSCOPICAL   EXAMINATION  OF  BACTERIA 


73 


Neutral  red,  i  per  cent,  aqueous  solution. 
Leishman's  modified  Romano wsky  stain. 
Carbolic  acid,  5  per  cent,  aqueous  solution. 


FIG.  65. — Staining  rack,  rubber  change  mat  and  lysol  pot 

Acetic  acid,  i  per  cent,  solution. 
Sulphuric  acid,  25  per  cent,  solution. 
Xylol. 


FIG.  66.— Drop-bottle. 


FIG.  67. — Canada  balsam  pot. 


And  two  pots  with  air-tight  glass  caps  (Fig.  67),  each 
provided  with  a  piece  of  glass  rod  and  filled  respec- 


74  MICROSCOPICAL   EXAMINATION    OF   BACTERIA 

lively  with  Canada  balsam  dissolved  in  xylol,  and  sterile 
vaseline. 

METHODS  OF  EXAMINATION. 

Bacteria,  etc.,  are  examined  microscopically. 

1.  In  the  living  state,  unstained,  or  stained. 

2.  In  the  "fixed"  condition  (i.  e.,  fixed,  killed, 
and  stained  by  suitable  methods) . 

The  preparation  of  a  specimen  from  a  tube  cultiva- 
tion for  examination  by  these  methods  may  be  de- 
scribed as  follows : 

1.  Living,  Unstained. — (a)  "Fresh"  Preparation. — 
i.  Clean  and  dry  a  3  by  i  glass  slip  and  place  it  on 
one  of  the  squares  of  filter  paper.  Deposit  a  drop  of 
water  (preferably  distilled)  or  a  drop  of  i  per  cent, 
solution  of  caustic  potash,  on  the  centre  of  the  slip, 
by  means  of  the  platinum  loop. 


5" 


&  a 

B  fc 
u  < 
w 

p 


H 


FIG.  68. — Holding  tubes  for  removing  bacterial  growth, 
as  seen  from  the  front. 

2.  Remove  the  tube  cultivation  from  its 
rack  or  jar  with  the  left  hand  and  ignite  the 
cotton- wool  plug  by  holding  it  to  the  flame  of 
the  Bunsen  burner.  Extinguish  the  flame  by 
blowing  on  the  plug,  whilst  rotating  the  tube 
on  its  long  axis,  its  mouth  directed  vertically 
upward,  between  the  thumb  and  fingers.  (This 
operation  is  termed  "flaming  the  plug,"  and  is 


LIVING,    UNSTAINED  75 

intended  to  destroy  any  micro-organisms  that 
may  have  become  entangled  in  the  loose  fibres 
of  the  cotton-wool,  and  which,  if  not  thus  de- 
stroyed, might  fall  into  the  tube  when  the  plug 
is  removed  and  so  accidentally  contaminate  the 
cultivation.) 

3 .  Hold  the  tube  at  or  near  its  centre  between 
the  ends  of  the  thumb  and  first  two  ringers  of 
the  left  hand,  and  allow  the  sealed  end  to  rest 
upon  the  back  of  the  hand  between  the  thumb 

5  and  forefinger,  the  plug  pointing  to  the  right. 
Keep  the  tube  as  nearly  in  the  horizontal  posi- 
tion as  is  consistent  with  safety,  to  diminish 
the  risk  of  the  accidental  entry  of  organisms 
(Fig.  68). 

4.  Take  the  handle  of  the  loop  between  the 
thumb  and  forefinger  of  the  right  hand,  holding 
the  instrument  in  a  position  similar  to  that 
occupied  by  a  pen  or  a  paint-brush,  and  sterilise 
the  platinum  portion  by  holding  it  in  the  flame 
of  a  Bunsen  burner  until  it  is  red  hot.     Sterilise 
the  adjacent  portion  of  the  aluminium  handle 
by  passing  it  rapidly  twice  or  thrice  through 
the  flame.     After  sterilising  it,  the  loop  must 
not  be  allowed  to  leave  the  hand  or  to  touch 
against  anything  but  the  material  it  is  intended 
to  examine,  until  it  is  finished  with  and  has  been 
again  sterilised. 

5.  Grasp  the  cotton-wool  plug  of  the  test- 
tube  between  the  little  finger  and  the  palm  of 
the  right  hand  (whilst  still  holding  the  loop  as 
directed  in  step   4),   and  remove  it  from  the 
mouth  of  the  tube  by  a  "  sere  wing"  motion  of 
the  right  hand. 

6.  Introduce  the  platinum  loop  into  the  tube 
and  hold  it  in  this  position  until  satisfied  that 

i  it  is  quite  cool.     (The  cooling  may  be  hastened 


76  MICROSCOPICAL   EXAMINATION    OF    BACTERIA 


by  touching  the  loop  on  one  of  the  drops  of 
moisture  which  are  usually  to  be  found  con- 
densed on  the  interior  of  the  glass  tube,  or  by 
dipping  it  into  the  condensation  water  at  the 
bottom;  at  the  same  time  care  must  be  taken 
in  the  case  of  cultures  on  solid  media  to  avoid 
touching  either  the  medium  or  the  growth.) 

7.  Remove  a  small  portion  of  the  growth  by 
taking  up  a  drop  of  liquid,  in  the  case  of  a  fluid 
culture,  in  the  loop ;  or  by  touching  the  loop  on 
the  surface  of  the  growth  when  the  culture  is  on 
solid  medium;  and  withdraw  the  loop  from  the 
tube  without  again  touching  the  medium  or 
the  glass  sides  of  the  tube. 

8.  Replace    the    cotton-wool    plug    in    the 
mouth  of  the  tube. 

9.  Replace  the  tube  cultivation  in  its  rack  or  jar. 

10.  Mix  the  contents  of  the  loop  thoroughly  with 
the  drop  of  water  on  the  3  by  i  slide. 

11.  Again  sterilise  the  loop  as  directed  in  step  4, 
and  replace  it  in  its  stand. 

12.  Remove  a  cover-slip  from  the  glass  capsule  by 
means  of  the  cover-slip  forceps,  rest  it  for  a  moment 
on  its  edge,  on  a  piece  of  filter  paper  to  remove  the 
excess  of  alcohol,  then  pass  it  through  the  flame  of  the 
Bunsen  burner.     This  burns  off  the  remainder  of  the 
alcohol,  and  the  cover-slip  so  "flamed"  is  now  clean, 
dry,  and  sterile. 

13.  Lower  the  cover-slip,  still  held  in  the  forceps, 
on  to  the  surface  of  the  drop  of  fluid  on  the  3  by  i 
slip,  carefully  and  gently,  to  avoid  the  inclusion  of  air 
bubbles. 

14.  Examine  microscopically  (vide  infra) . 

During  the  microscopical  examination,  stains  and 
other  reagents  may  be  run  in  under  a  cover-slip  by 
the  simple  method  of  placing  a  drop  of  the  reagent  in 
contact  with  one  edge  of  the  cover-glass  and  apply- 


BLACK   AND    WHITE    FILMS  77 

ing  the  torn  edge  of  a  piece  of  blotting  paper  to  the 
opposite  side.  The  reagent  may  then  be  observed 
to  flow  across  the  field  and  come  into  contact  with 
such  of  the  micro-organisms  as  lie  in  its  path. 

The  non-toxic  basic  dyes  most  generally  employed 
for  the  intra-vitam  staining  of  bacteria  are 


Neutral  red, 
Quinoleine  blue 
Methylene  green 
Vesuvin, 


in  0.5  per  cent,  aqueous  solutions. 


Negative  Stain  (Burri). — By  this  method  of  demon- 
stration the  appearances  presented  by  dark  ground 
illumination  (by  means  of  a  paraboloid  condenser) 
are  closely  simulated,  since  minute  particles,  bacteria, 
blood  or  pus  cells  etc.  stand  out  as  brilliantly  white  or 
colourless  bodies  on  a  dark  grey-brown  background. 

Reagent  required: 

Any  one  of  the  liquid  waterproof  black  drawing  inks 
(Chin-chin,  Pelican,  etc.) .     This  is  prepared  for  use  as 
follows : 
Measure  out  and  mix : 

Liquid  black  ink, 25  c.c. 

Tincture  of  iodine i  c.c. 

Allow  the  mixture  to  stand  24  hours,  centrifugalise 
thoroughly,  pipette  off  the  supernatant  liquid  to  a  clean 
bottle  and  then  add  a  crystal  of  thymol  or  one  drop  of 
formalin  as  a  preservative. 

METHOD.— 

1.  With  the  sterilised  loop  deposit  one  drop  of  the 
liquid  ink  close  to  one  end  of  a  3  by  i  slide. 

2.  With  the  sterilised  loop  deposit  a  drop  of  the 
fluid  culture  (or  of  an  emulsion  from  a  solid  culture) 
by  the  side  of  the  drop  of  ink  (Fig.  69,  a) ;  mix  the  two 
drops  thoroughly  by  the  aid  of  the  loop. 

3.  Sterilise  the  loop. 


78  MICROSCOPICAL   EXAMINATION    OF   BACTERIA 

4.  Hold  the  slide  firmly  on  the  bench  with  the  thumb 
and  forefinger  of  the  left  hand  applied  to  the  end 
nearest  the  drop  of  fluid. 

5.  Take  another  clean  3  by  i  slide  in  the  right  hand 
and  lower  its  short  end  obliquely  (at  an  angle  of  about 
60°)  transversely  on  to  the  mixed  ink  and  culture  en 
the  first  slide,  and  allow  the  fluid  to  spread  across  the 
slide  and  fill  the  angle  of  incidence. 

6.  Maintaining  the  original  angle,  draw  the  second 
slide  firmly  and  evenly  along  the  first  toward  the  end 
farthest  from  the  left  hand  (Fig.  69,  b). 

7.  Throw  the  second  slide  into  a  pot  of  disinfectant; 
allow  the  first  slide  to  dry  in  the  air. 


FIG.  69. — Spreading  negative  film. 

8.  Place  a  drop  of  immersion  oil  on  the  centre  of  the 
film,  lower  the  i/i 2-inch  objective  into  the  oil  and 
examine  microscopically  without  the  intervention  of 
a  cover-slip. 

(The  film  of  ink  may  be  covered  with  a  long  cover- 
glass  and  xylol  balsam  as  a  permanent  preparation.) 

(b)  Hanging-drop  Preparation. — 

1.  Smear  a  layer  of  sterile  vaseline  on  the  upper 
surface  of  the  ring  cell  of  a  hanging-drop  slide  by  means 
of  the  glass  rod  provided  with  the  vaseline  bottle,  and 
place  the  slide  on  a  piece  of  filter  paper. 

2.  "Flame"  a  cover-slip  and  place  it  on  the  filter 
paper  by  the  side  of  the  hanging-drop  slide. 

3.  Place  a  drop  of  water  on  the  centre  of  the  cover- 
slip  by  means  of  the  platinum  loop. 


HANGING    DROP  79 

4.  Obtain  a  small  quantity  of  the  material  it  is 
desired  to  examine,  in  the  manner  detailed  above  (pages 
74-76,  steps  2  to  ii  must  be  followed  in  their  entirety 
and  with  the  strictest  exactitude  whenever  tube  contents 
are  being  handled),  and  mix  it  with  the  drop  of  water 
on  the  cover-slip. 

5.  Raise  the  cover-slip  in  the  points  of  the  forceps 
and  rapidly  invert  it  on  to  the  ring  cell  of  the  hanging- 
drop  slide,  so  that  the  drop  of  fluid  occupies  the  centre 
of  the  ring.     (Carefully  avoid  contact   between  the 
drop  of  fluid  and  either  the  ring  cell  or  the  layer  of 
vaseline.     Should  this  happen,  the  now  infected  hang- 
ing-drop slide  and  its  cover-slip  must  be  dropped  into 
the  pot  of  lysol  and  a  new  preparation  made.) 

6.  Press  the  cover-slip  firmly  down  into  the  vaseline 
on  to  the  top  of  the  ring  cell.     (This  spreads  out  the 
vaseline  into  a  thin  layer,  and  besides  ensuring  the 
adhesion  of  the  cover-slip,  seals  the  cells  and  so  retards 
evaporation.) 

7.  Examine  microscopically. 

The  examination  of  a  " fresh"  specimen  or  a  " hang- 
ing-drop" preparation  is  directed  to  the  determination 
of  the  following  data : 

1.  The  nature  of  the  bacteria  present — e.  g.,  cocci, 
bacilli,  etc. 

2.  The  purity  of  the  cultivation;  this  can  only  be 
determined  when  gross  morphological  differences  exist 
between  the  organisms  present. 

3 .  The  presence  or  absence  of  spores ;  when  present, 
spores   show  their  typical  refrangibility  exceedingly 
well  by  this  method. 

4.  The  presence  or  absence  of  mobility.     In  a  hang- 
ing-drop specimen  some  form  of  movement  can  prac- 
tically always  be  observed,  and  its  character  must  be 
carefully  determined  by  noting  the  relative  positions 
of  adjacent  micro-organisms. 

(a)    Brownian  or  molecular  movement.     Minute  par- 


80  MICROSCOPICAL   EXAMINATION    OF   BACTERIA 

tides  of  solid  matter  (including  bacteria),  when  sus- 
pended in  a  fluid,  will  always  show  a  vibratory  move- 
ment affecting  the  entire  field,  but  never  altering  the 
relative  positions  of  the  bacteria.  (Cocci  exhibit  this 
movement,  but  with  the  exception  of  the  Micrococcus 
agilis,  the  cocci  are  non-motile.) 

(b)  Streaming  movement.     This  is  due  to  currents 
set  up  in  the  hanging  drop  as  a  result  of  jarring  of  the 
specimen  or  of  evaporation,  or  to  the  fact  that  the 
cover-slip   is   not   perfectly   level,    and   although   the 
relative  positions  of  the  bacteria  may  vary,  still  the 
flowing  movement  of  large  numbers  of  organisms  in 
some  one  direction  will  usually  be  sufficient  to  demon- 
strate the  nature  of  this  motion. 

(c)  Locomotive  movement,  or  true  motility,  is  deter- 
mined by  observing  some  one  particular  bacillus  chang- 
ing its  position  in  the  field  independently  of,  and  in  a 
direction  contrary  to,  other  organisms  present. 

When  the  examination  is  completed  and  the  specimen 
finished  with,  the  "  fresh  specimen  " — i.  e.,  the  slide  with 
the  cover-slip  attached — must  be  dropped  into  the 
lysol  pot.  In  the  hanging-drop  specimen,  however, 
the  cover-slip  only  is  infected,  and  this  may  be  raised 
from  the  ring  cell  by  means  of  forceps  and  dropped 
into  the  disinfectant. 

Permanent  Staining  of  the  Hanging-drop  Specimen. — 
Occasionally  it  is  necessary  to  fix  and  stain  a  hanging- 
drop  preparation.  This  may  be  done  as  follows : 

1.  Remove  the  cover-slip  from  the  cell  by  the  aid  of 
the  forceps. 

2.  If  the  drop  is  small,  fix  it  by  dropping  it  face 
downward,  whilst  still  wet,  on  to  the  surface  of  some 
Gulland  's  solution  or  corrosive  sublimate  solution  (vide 
page  82)  in  a  watch-glass.     If  the  drop  is  large,  place 
it  face  upward  on  the  rubber  mat,  cover  it  with  an 
inverted  watch-glass,  and  allow  it  to  dry.     Then  fix 
it  in  the  alcohol  and  ether  solution  (vide,  page  82) . 


KILLED,    STAESfED  8 1 

3.  Dip  the  cover-glass  into  a  beaker  containing  hot 
water  in  order  to  remove  some  of  the  vaseline  adhering 
to  it. 

4.  Wash  successively  in  alcohol,   xylol,  ether,  and 
alcohol,  to  remove  the  last  traces  of  grease. 

5.  Wash  in  water. 

6.  Stain,  wash,  dry,  and  mount  as  for  an  ordinary 
cover-slip  film  preparation  (vide  pages  83-85). 

2.  Killed,  Stained. — In  this  method  three  distinct 
processes  are  necessary: 

"Preparing"  and  "fixing"  the  film. 

Staining. 

Mounting. 

Preparing  the  Film. — 

1.  Flame  a  cover- slip  and  place  is  on  a  piece  of  filter 
paper. 

2.  Place  a  drop  of  water  on  the  centre  of  the  cover- 
slip  by  means  of  platinum  loop. 

3.  Obtain  a  small  quantity  of  the  material   to  be 
examined  upon  a  sterilised  platinum  loop  (see  pages 
74-76,  steps  2  to  u)  and  mix  it  with  the  drops  of  water 
on  the  cover-slip. 

4.  Spread  the  drop   of  emulsion  evenly  over  the 
cover-slip  in  the  form  of  a  square  film  to  within  i  mm. 
of  each  edge  of  the  cover-slip. 

5.  Allow  it  to  dry  completely  in  the  air. 

Fixing. — Fix  by  passing  the  cover-slip,  held  in  the 
fingers,  three  or  four  times  through  the  flame  of  a  Bun- 
sen  burner. 

In  some  instances  (e.  g.,  when  the  films  after  staining 
are  intended  for  micrometric  observations)  it  is  almost 
essential  to  fix  by  exposure  to  a  uniform  temperature 
of  115°  C.,  for  twenty  minutes.  This  is  best  done  in  a 
carefully  regulated  hot-air  oven. 

Fixation  may  also  be  effected  by  immersing  in  some 
fixative  fluid,  such  as  one  of  the  following: 
6 


82  MICROSCOPICAL   EXAMINATION    OF   BACTERIA 

1.  Absolute  alcohol,  for  five  to  fifteen  minutes. 

f  equal  parts,  for  five  to  thirty 

2.  Absolute  alcohol,  .  ,          r      ui     j 

_  ,  <     minutes  (e.  g.,  for  blood  or 

.bther,  M1  \ 

[     milk) . 

3.  Osmic  acid,    i   per  cent,   aqueous   solution,    for 
thirty  seconds. 

4.  Corrosive  sublimate,  saturated  aqueous  solution, 
for  five  minutes. 

5.  Corrosive    sublimate    (Lang),    for    five    minutes. 
This  solution  is  prepared  by  dissolving: 

Sodium  chloride o .  7  5  gramme 

Hydrarg.  perchloride 12.00  grammes 

Acetic  acid 5  .  oo  grammes 

In  distilled  water loo.ooc.c. 

Filter. 

6.  Gulland's  solution,  for  five  minutes.     This  solu- 
tion is  prepared  by  mixing : 

Absolute  alcohol 25.oc.c. 

Ether 25.oc.c. 

Corrosive  sublimate,   20  per  cent,  al- 
coholic solution 0.4  c.c. 

7 .  Formalin  i  o  per  cent .  aqueous  solution  ( =  4  per  cent, 
aqueous  solution  of  formaldehyde  since  formalin  is  a  40 
per  cent,  solution  of  the  gas  in  water) . 

Either  of  these  methods  of  fixation  coagulates  the 
albuminous  material  and  ensures  perfect  adhesion  of 
the  film  to  the  cover-slip. 

Clearing. — Wash  the  cover-slip  thoroughly  in  running 
water  and  proceed  with  the  staining. 

If  the  film  has  been  prepared  from  broth,  liquefied 
gelatine,  or  pus  or  other  morbid  exudations,  saturate 
the  film  after  fixation  with  acetic  acid  2  per  cent,  and 
allow  it  to  act  for  two  minutes. 

Wash  with  alcohol,  then  let  the  alcohol  remain  on 
the  cover-slip  for  two  minutes.  (This  will  "  clear"  the 
groundwork  and  give  a  much  sharper  and  cleaner  film 
than  would  otherwise  be  obtained.) 


KILLED,    STAINED  83 

If  the  film  has  been  prepared  from  blood  or  blood- 
stained fluid,  treat  with  acetic  acid  2  per  cent,  for  two 
minutes  after  fixation.  Wash  with  water,  dry,  and 
proceed  with  the  staining.  (This  will  remove  the 
haemoglobin  and  facilitate  examination.) 

Staining. — 

1.  Rest  the  cover-slip,  film  side  uppermost,  on  the 
rubber  mat. 

2.  By  means  of  a  drop-bottle,  cover  the  film  side  of 
the  cover-slip  with  the  selected  stain,  allow  it  to  act 
for  a  few  minutes,  then  wash  off  the  excess  in  running 
water. 

The  penetrating  power  of  stains  is  increased  by  (a) 
physical  means — e.  g.,  heating  the  stain;  (b)  chemical 
means — e.  g.,  by  the  additon  of  carbolic  acid,  5  per 
cent,  aqueous  solution;  caustic  alkalies,  2  per  cent, 
aqueous  solutions;  water  saturated  with  aniline  oil; 
borax,  0.5  per  cent,  aqueous  solution. 

The  most  commonly  used  dyes  for  cover-slip  film 
preparations  are  the  aniline  dyes. 
(A)   Basic: 

(a)  Methylene-blue. 

(b)  Gentian  violet. 

(c)  Fuchsin. 

These  dyes  are  kept  in  saturated  alcoholic  (90  per 
cent.)  solutions  so  that  decomposition  may  be  retarded. 

Two  or  three  drops  of  alcoholic  solution  of  these 
dyes  to,  say,  4  c.c.  water,  usually  makes  a  sufficiently 
strong  staining  fluid  for  cover-slip  film  preparations. 

Carbolic  methylene-blue  (C.M.B.)  and  carbol  f uchsin 
(C.F.)  are  prepared  by  covering  the  cover-slip  with  5 
per  cent,  solution  of  carbolic  acid  and  adding  a  few 
drops  of  the  saturated  alcoholic  solution  of  methylene- 
blue  or  fuchsin  respectively  to  it.  For  aniline  gentian 
violet  (A.G.V.)  the  stain  is  added  to  a  saturated  solu- 
tion of  aniline  oil  in  water. 


84  MICROSCOPICAL   EXAMINATION    OF   BACTERIA 

(d)  Thionin  blue. 

(e)  Bismarck  brown. 

(f)  Neutral  red. 
(B)  Acid: 

(a)  Eosin,  aqueous  yellowish. 

(b)  Safranine. 

These  dyes  are  kept  in  i  per  cent,  aqueous  solution 
to  which  is  added  5  per  cent,  of  alcohol,  as  a  preserva- 
tive. They  are  generally  used  in  this  form. 

A  few  nuclear  stains  (carmine,  haematoxylin)  are 
occasionally  used  more  especially  in  " section"  work. 

Decolonisation. — After  overstating,  films  may  be 
decolourised  by  washing  for  a  longer  or  shorter  time 
in  one  of  the  following  reagents  arranged  in  ascending 
order  of  power 

1.  Water. 

2.  Chloroform. 

3.  Acetic  acid,  i  per  cent. 

4.  Alcohol. 

5.  Alcohol  absolute,  \ 

.     , .       . ,  }  equal  parts. 

Acetic  acid,  i  percent.,  J 

Hydrochloric,  i  per  cent,  aqueous 

solution. 

Hydrochloric,    i   per  cent,   alco- 
holic.   (90  per  cent.)     solution. 
Sulphuric,  25    per  cent,  aqueous 

solution. 

Nitric,  33  per  cent,  aqueous  solu- 
tion. 

Counter  staining. — Use   colours   which   will   contrast 
with  the  first  stain;  e.  g., 
Vesuvin, 


6.  Mineral  acids : 


for  films  stained  by  methylene-blue  or 
Gram's  method. 


Neutral    red, 

Eosin, 

Fuchsin, 

Methylene-blue,     )  £       ^  .   •     j     «        r    u  • 

~       .         .  ,  }  for    films    stained    by    fuchsin. 

Gentian  violet, 


IMPRESSION    FILMS  85 

8.  Mounting. — 

1.  Wash  the  film  carefully  in  running  water. 

2.  Blot   off  the   superfluous   water  with  the   filter 
paper,  or  dry  more  completely  between  two  folds  of 
blotting  paper. 

3.  Complete  the  drying  in  the  air,  or  by  holding  the 
cover-slip  in  the  fingers  at  a  safe  distance  above  the 
flame  of  the  Bunsen  burner. 

4.  Place  a  drop  of  xylol  balsam  on  the  centre  of  a 
clean  3  by  i  glass  slide  and  invert  the  cover-slip  over 
the  balsam,  and  lower  it  carefully  to  avoid  the  inclusion 
of  air  bubbles. 

NOTE. — Xylol  is  used  in  preference  to  chloroform  to  dissolve 
Canada  balsam,  as  it  does  not  decolourise  the  specimen. 

Impression  films  (Klatschpraeparat)  are  prepared 
from  isolated  colonies  of  bacteria  in  order  that  their 
characteristic  formation  may  be  examined  by  higher 
powers  than  can  be  brought  to  bear  on  the  living  culti- 
vation. They  are  prepared  from  plate  cultivations 
(vide  page  230)  in  the  following  manner. 

1 .  Remove  a  clean  cover-slip  from  the  alcohol  pot 
with  sterile  forceps  and  burn  off  the  spirit. 

2.  Open  the  plate  and  rest  one  edge  of  the  cover- 
slip  on  the  surface  of  the  medium  a  little  to  one  side 
of  the  selected  colony.     Lower  it  cautiously  over  the 
colony  until  horizontal.     Avoid  any  lateral  movement 
or  the  inclusion  of  bubbles  of  air. 

3.  Make  gentle  vertical  pressure  on  the  centre  of  the 
cover-slip  with  the  points  of  the  forceps  to  ensure 
perfect  contact  with  the  colony. 

4.  Steady  one  edge  of  the  cover-slip  with  the  forceps 
and  pass  the  point  of  a  mounted  needle  just  under 
the  opposite  edge  and  raise  the  cover-slip  carefully; 
the  colony  will  be  adherent  to  it.     When  nearly  verti- 
cal, grasp  the  cover-slip  with  the  forceps  and  remove 
it  from  the  plate.     Re-cover  the  plate. 

5.  Place  the  cover-slip,  film  uppermost,  on  the  rubber 


86  MICROSCOPICAL    EXAMINATION    OF    BACTERIA 

mat,  and  cover  it  with  an  inverted  watch-glass  until 
dry. 

6.  Fix  by  immersing  in  one  of  the  fixing  fluids  pre- 
viously mentioned  (vide  page  82). 

7.  Clear  with  acetic  acid  and  alcohol.  ' 

8.  Stain  and  mount  as  an  ordinary  cover-slip  film 
preparation,  being  careful  to  perform  all  washing  op- 
erations with  extreme  gentleness. 

Microscopical  Examination  of  the  Unstained  Speci= 
mens. — 

1.  Place  the  body  tube  of  the  microscope  in  the  ver- 
tical position. 

2.  Arrange  the  hanging-drop   slide  on   the   micro- 
scope stage  so  that  the  drop  of  fluid  is  in  the  optical 
axis  of  the  instrument,  and  secure  it  in  that  position 
by  means  of  the  spring  clips. 

3.  Use  the  J-inch  objective,   rack  down  the  body 
tube  until  the  front  lens  of  the  objective  is  almost  in 
contact  with  the  cover-slip — that  is,  well  within  its 
focal  distance.     This  is  best  done  whilst  bending  down 
the  head  to  one  side  of  the  microscope,  so  that  the 
eyes  are  on  a  level  with  the  stage. 

4.  Apply  the  eye  to  the  ocular  and  adjust  the  plane 
mirror  to  the  position  which  secures  the  best  illumina- 
tion. 

5.  Rack  the  condenser  down  slightly  and  cut  down 
the  aperture  of  the  iris  diaphragm  so  that  the  light, 
although  even,  is  dim. 

6.  Rack  up  the  body  tube  by  means  of  the  coarse 
adjustment  until  the  bacteria  come  into  view;  then 
focus  exactly  by  means  of  the  fine  adjustment. 

Some  difficulty  is  often  experienced  at  first  in  finding 
the  hanging  drop,  and  if  the  first  attempt  is  unsuccess- 
ful, the  student  must  not  on  any  account,  whilst  still 
applying  his  eye  to  the  ocular,  rack  the  body  tube 
down  (for  by  so  doing  there  is  every  likelihood  of  the 


DARK    GROUND    ILLUMINATION  87 

front  lens  of  the  objective  being  forced  through  the 
cover-glass,  and  not  only  spoiling  the  specimen,  but  also 
contaminating  the  objective) ;  but,  on  the  contrary, 
withdraw  his  eye,  rack  the  tube  up,  and  commence 
again  from  step  2. 

Dark  Ground  Illumination.— 

1 .  Set  up  the  microscope  stand  in  the  vertical  position 
and  insert  the  highest  eyepiece  available. 

2.  Remove  the  nosepiece  from  the  microscope  tube 
and  fit  the  f  inch  objective  in  place. 

3.  Remove  the  substage  condenser  and  replace  it  by 
the  dark  ground  condenser. 

4.  Fit  up  the  source  of  illumination  some  30-50  cm. 
distant  from  the  microscope.     (This  should  be  the  Lili- 
put  Arc  Lamp  (Leitz) ,  Nernst  Lamp  or  incandescent 
gas  lamp ;  if  either  of  the  two  latter  are  employed,  a  bull's 
eye  condenser  to  produce  parallel  rays  must  be  inter- 
posed between  light  and  microscope) ;  and  adjust  illumi- 
nant  and  microscope  so  that  the  substage  plane  mirror  is 
completely  filled  with  light. 

5.  Focus  the  two  concentric  rings  engraved  upon  the 
upper  surface  of  the  condenser  and  centre  them  accu- 
rately by  means  of  the  centring  screws. 

6.  Prepare  a  "fresh"  specimen  (see  pages  74-76) of  tne 
material  it  is  desired  to  observe,  using  selected,  new, 
3  by  i  glass  slips  of  less  than  i  mm.  thickness,  and  No. 
i  cover-glasses    (0.17    mm.    thick),    which   should    be 
cleaned  with  a  piece  of  soft  washleather  and  not  with  the 
emery  paper,  as  scratches  on  the  glass  produce  haziness 
in  the  preparation). 

7.  Deposit  a  large  drop  of  immersion  oil  (or  pure 
water)  on  the  upper  surface  of  the  condenser  and  rack  it 
down  a  few  millimetres. 

8.  Adjust  the  fresh  preparation  on  the  microscope 
stage  and  fasten  it  in  position  with  the  stage  clips. 

9.  Rack    up    the    condenser    until    the    immersion 


88  MICROSCOPICAL   EXAMINATION    OF   BACTERIA 

fluid  makes  contact  with  the  tinder  surface  of  the  slide ; 
avoid  the  formation  of  air  bubbles. 

10.  Adjust  the  substage  mirror  so  that  the  light  is 
reflected  upward.     A  bright  spot  will  be  seen  on  the 
fresh  preparation  near  the  centre  of  the  field. 

11.  Replace   the  §-inch  objective    by    the   TV-inch 
oil  immersion  lens  which  has  been  fitted  with  the  special 
stop  to  reduce  its  N.  A. ;  place  a  drop  of  immersion  oil 
upon  the  centre  of  the  cover-glasses  of  the  fresh  prepa- 
ration and  lower  the  microscope  tube  until  the  front 
lens  of  the  objective  has  entered  the  oil  drop. 

12.  Focus  the  bright  spot  referred  to 
in  step  10.     If  it  no  longer  occupies  the 
centre  of  the  field,  alter  the  angle  of  the 
sub-stage  mirror  until  it  does. 

13.  Now  focus  the  lens  accurately  on 
the  film,  cautiously  vary  the  height  of 
the  dark  ground  condenser  until  the  best 
position  is  found.     The  intensely  illumi- 
nated bacteria  will  stand  out  in  vivid 
contrast  to  the  dark  background. 

FIG.  70.— immer-       Microscopical   Examination    of    the 

sion  oil  bottle. 

Stained  Specimen. — (The  body  tube  of 
the  microscope  may  be  vertical  or  inclined  to  an 
angle.) 

1.  Secure  the  slide  on  the  stage  of  the  microscope  by 
means  of  the  spring  clips. 

2.  Place  a  drop  of  cedarwood  oil  on  the  centre  of  the 
cover-slip. 

The  immersion  oil  is  pure  cedarwood  oil,  and  is  kept  in  a  small 
bottle  of  stout  glass  (Fig.  70) ,  the  cavity  of  which  is  shaped  like  an 
inverted  cone,  and  is  provided  with  a  safety  funnel  (so  that  the  oil 
does  not  escape  if  the  bottle  is  accidently  overturned)  and  a  dust 
cap  of  boxwood  fitted  with  a  wooden  rod  with  which  the  drop  of 
oil  is  applied  to  the  coverglass  or  lens. 

3.  Use  the  Ty-inch  oil  immersion  lens  of  the  micro- 
scope.    Rack  down  the  body  tube  till  the  front  lens 


STAINED    FILMS  89 

of  the  objective  is  in  contact  with  the  oil  and  nearly 
touching  the  cover-slip. 

4.  Rack  up  the  condenser  until  it  is  in  contact  with 
the  under  surface  of  the  slide. 

5.  Apply  the  eye  to  the  ocular  and  arrange  the  plane 
mirror  so  as  to  obtain  the  greatest  possible  amount  of 
light. 

6.  Rack  up  the  body  tube   until   the   stained  film 
comes  into  view. 

7.  Focus  the  condenser  accurately  on  the  film. 

8.  Focus  the  film  accurately  by  means  of  the  fine 
adjustment. 


VI.  STAINING  METHODS. 

IN  the  following  pages  are  collected  the  various 
"stock"  stains  in  everyday  use  in  the  bacteriological 
laboratory,  together  with  a  selection  of  the  most  con- 
venient and  generally  useful  staining  methods  for 
demonstrating  particular  structures  or  differentiating 
groups  of  bacteria.  The  stains  employed  should  either 
be  those  prepared  by  Gruebler,  of  Leipzig,  or  Merck,  of 
Darmstadt.  The  methods  printed  in  ordinary  type 
are  those  which  a  long  experience  has  shown  to  be  the 
most  reliable,  and  to  give  the  best  results — those 
relegated  to  small  type  comprise  such  as  are  not  so 
generally  useful,  but  give  excellent  results  in  the  hands 
of  the  experienced  worker. 

BACTERIA  STAINS. 

Methylene-blue.— 

1.  Saturated  Aqueous  Solution. 
Weigh  out 

Methylene-blue 1.5  grammes 

Place  in  a  stoppered  bottle  having  a  capacity  of 
from  150  to  200  c.c.  and  add 

Distilled  water 100.0  c.c. 

Allow  the  water  to  remain  in  contact  with  the  dye 
for  two  weeks,  shaking  the  contents  of  the  bottle  vig- 
ourously  for  a  few  moments  every  day.  Filter. 

2.  Saturated  Alcoholic  Solution. 
Weigh  out 

Methylene-blue 1.5  grammes 

90 


SIMPLE    STAINS  9 1 

Place  in  a  stoppered  bottle  of  150  c.c.  capacity  and 
add 

Alcohol,  90  per  cent 100.0  c.c. 

Allow  the  alcohol  to  remain  in  contact  with  the  dye 
for  two  hours,  shaking  vigourously  every  few  minutes. 
Filter. 

3.  Carbolic  Methylene-blue  (Kuehne). 
Weigh  out 

Methylene-blue 1.5  grammes 

Carbolic  acid 5.0  grammes 

and  dissolve  in 

Distilled  water 100.0  c.c. 

and  add 

Absolute  alcohol 10.0   c.c. 

Filter. 

4.  Alkaline  Methylene-blue  (Loeffler). 
Measure  out  and  mix 

Methylene-blue,  saturated  alcoholic  solution.  .    .      30.0  c.c. 
Caustic  potash,  o.oi  per  cent,  aqueous  solution  .    100.0  c.c. 

Filter. 

Gentian  Violet.— 

5.  Saturated  Aqueous  Solution. 
Weigh  out 

Gentian  violet 2.25   grammes 

and  proceed  as  in  preparing  the  corresponding  solution 
of  methylene-blue. 

6.  Saturated  Alcoholic  Solution. 
Weigh  out 

Gentian  violet 5.0   grammes 

and  proceed  as  in  preparing  the  corresponding  solution 
of  methylene-blue. 


£2  STAINING   METHODS 

7.  Carbolic  Gentian  Violet  (Nicolle). 
Measure  out  and  mix 

Gentian  violet,  saturated  alcoholic  solution    .      10.0   c.c. 
Carbolic  acid,  i  per  cent,  aqueous  solution    .    100.0   c.c. 

Filter. 

8.  Anilin  Water  Solution  (Koch-Ehrlich) . 
Measure  out 

Distilled  water 100  c.c. 

Add  anilin  oil  drop  by  drop  (shaking  well  after  the 
addition  of  each  drop)  until  the  solution  is  opaque. 

Filter  until  clear, 
and  add 

Absolute  alcohol 10  c.c. 

Saturated  alcoholic  solution  gentian  violet    .    1 1  c.c. 

Filter. 

NOTE. — This  solution  will  not  keep  longer  than  14  days. 

Thionine  Blue  (or  Lauth's  Violet).— 

9.  Carbolic  Thionine  Blue  (Nicolle). 
Weigh  out 

Thionine  blue i .  o    gramme 

Carbolic  acid 2.5    grammes 

and  dissolve  in 

Distilled  water 100.0   c.c. 

Filter. 

Before  use  dilute  with  equal  quantity  of  distilled 
water  and  again  filter. 

Fuchsin  (Basic).— 

10.  Saturated  Aqueous  Solution. 
Weigh  out 

Basic  fuchsin 1.5  grammes 

and  proceed  as  in  preparing  the  corresponding  solution 
of  methylene-blue  (q.  v.) . 


CONTRAST    STAINS  93 

11.  Saturated  Alcoholic  Solution. 
Weigh  out 

Basic  fuchsin 3.5    grammes 

and  proceed  as  in  preparing  the  corresponding  solu- 
tion of  methylene-blue. 

1 2 .  Carbolic  Fuchsin  (Ziehl) . 
Weigh  out 

Basic  fuchsin i .  o    gramme 

Carbolic  acid 5.0    grammes 

dissolve  in 

Distilled  water 100.0   c.c. 

and  add 

Absolute  alcohol 10.0    c.c. 

Filter. 

CONTRAST  STAINS. 

Eosin. — There  are  several  commercial  varieties  of 
eosin,  which,  from  the  bacteriological  point  of  view, 
possess  very  different  values.  Gruebler  lists  four  varie- 
ties, of  which  two  only  are  useful  for  bacteriological 
work: 

Eosin,  aqueous  yellowish. 

Eosin,  aqueous  bluish. 

13.  Eosin  Aqueous  Solution    (Yellowish    or    Bluish 
Shade) ,  i  per  cent. 

Weigh  out 

Eosin,  aqueous i.o   gramme 

dissolve  in 

Distilled  water .    100.0   c.c. 

and  add 

Absolute  alcohol 5.0   c.c. 

Filter. 


94  STAINING   METHODS 

14.  Eosin  Alcoholic  Solution,  0.5  per  cent. 
Weigh  out 

Eosin,  alcoholic 0.5    gramme 

and  dissolve  in 

Alcohol  (70  per  cent.) 100.0   c.c. 

Filter. 

Safranine. — 

15.  A  queous  Solution . 
Weigh  out. 

Safranine 0.5    gramme 

and  dissolve  in 

Distilled  water    .........    100.0   c.c. 

Filter. 

Neutral  Red.— 

1 6.  Aqueous  Solution. 
Weigh  out 

Neutral  red i.o    gramme 

and  dissolve  in 

Distilled  water 100.0  c.c. 

Filter. 

Vesuvin  (or  Bismarck  Brown). — 

17.  Saturated  Aqueous  Solution. 
Weigh  out 

Vesuvin 0.5   gramme 

and  dissolve  in 

Distilled  water 100.0  c.c. 

Filter. 


H^EMATIN  95 

TISSUE  STAINS. 

Aniline  Gentian  Violet  (For  Weigert's  Fibrin  Stain)  .— 
Weigh  out 

Gentian  violet i .  o  gramme 

and  dissolve  in 

Absolute  alcohol 15.0  c.c. 

Distilled  water 80.0  c.c. 

then  add 

Aniline  oil 3.0  c.c. 

Shake  well  and  filter  before  use. 

Haematoxylin  (Ehrlich). — 

1.  Weigh  out 

Haematoxylin      2.0  grammes 

and  dissolve  in 

Absolute  alcohol 100.0  c.c. 

2.  Weigh  out 

Ammonium  alum 2.0   grammes 

and  dissolve  in 

Distilled  water loo.oc.c. 

3.  Mix  i  and  2,  allow  the  mixture  to  stand  forty- 
ight  hours,  then  filter. 

4.  Add 

Glycerine 85.0  c.c. 

Acetic  acid,  glacial lo.oc.c. 

5.  Allow  the  stain  to  stand  for  one  month  exposed 
to  light ;  then  filter  again  ready  for  use. 

Haematin  (Mayer's).— 
A.  Weigh  out 

Hsematin i.o  gramme 

and  dissolve  in 

Alcohol  90  per  cent,  (warmed  to  37°  C.)    .    50  c.c. 


96  STAINING   METHODS 

B.  Weigh  out 

Potash  alum 50  grammes 

and  dissolve  in 

Distilled  water 100  c.c. 

Prepare  these  two  solutions  in  separate  flasks. 
Take  a  clean  flask  of  250  c.c.  capacity  and  insert  a  large 
funnel  in  its  neck.  Pour  the  solutions  A  and  B  simul- 
taneously and  slowly  into  the  funnel  to  mix  thoroughly. 
Store  for  future  use. 

NOTE. — If -acid  haematin  is  required,  introduce  glacial  acetic 
acid  (3  c.c.)  into  the  mixing  flask  before  adding  the  solutions  A 
and  B. 

Alum  Carmine  (Mayer) . — 
Weigh  out 

Alum 2.5  grammes 

Carmine i .  o  gramme 

and  place  in  a  glass  beaker. 
Measure  out  in  a  measuring  cylinder, 

Distilled  water loo.oc.c. 

Place  the  beaker  on  a  sand-bath,  add  the  water  in 
successive  small  quantities,  and  keep  the  mixture  boil- 
ing for  twenty  minutes.  Measure  the  solution  and 
make  up  to  100  c.c.  by  the  addition  of  distilled  water. 
Filter. 

Lithium  Carmine  (Orth). — 
Weigh  out 

Carmine 2.5  grammes 

and  dissolve  in 

Lithium  carbonate,  cold  saturated  solution    .    100.0   c.c. 

Filter. 
Picrocarmine. — 

Weigh  out 

Picrocarmine 2.0  grammes 


BLOOD    STAIN  97 

and  dissolve  in 

Distilled  water 100.0  c.c. 

BLOOD  STAINS 

When  watery  solutions  of  medicinal  methylene  blue 
and  water  soluble  eosins  are  mixed  a  precipitate  is 
formed  which  is  soluble  only  in  alcohol,  and  solutions  of 
this  precipitate  .  impart  a  peculiar  reddish-purple 
colour  to  chromatin.  This  compound  was  first  used 
by  Romanowsky  to  demonstrate  malarial  parasites,  but 
various  modifications  are  now  employed  for  staining 
blood  films  generally,  and  also  for  bacteria  and  pro- 
tozoa. The  best  modifications  of  the  original  Roman- 
owsky are  those  of  Jenner  and  Leishman — Jenner 
being  most  suitable  for  the  histological  study  of  the 
blood,  and  Leishman  for  the  demonstration  of  protozoa. 

Jenner's  Stain.— 

A.  Weigh  out: 

Eosin  aqueous  yellow      ....6.0   grammes 

Dissolve  in 

Distilled  water  (non-alkaline)    .    .    250  c.c. 

This  will  make  a  thick  solution. 

B.  Weigh  out : 

Methylene  blue  (medicinally  pure)  Hoechst  .5.0  grammes 

Dissolve  in 

Distilled  water  (non-alkaline)       .    .    .    250  c.c. 

1.  Add  B  to  A  very  slowly,  stirring  all  the  time.     A 
viscous   precipitate  forms   which  frequently  loses  its 
viscosity  when   heat  is  applied.     (This   explains   the 
necessity  of  mixing  slowly) . 

2.  Evaporate  slowly  in  a  porcelain  basin,   stirring 
occasionally,  on  a  water  bath  at  55°  C.     When  a  paste 

7 


98  STAINING   METHODS 

begins   to   form   scrape    and    break   up    occasionally. 
(On  no  account  must  the  paste  be  allowed  to  fuse.) 

3.  Grind    the    resulting    mass    into    an    amorphous 
powder. 

4.  Weigh  out: 

Amorphous  powder 0.5  grammes 

Dissolve  in 

Methylic  alcohol  (Merck's  puriss.  for  analysis)    .    100  c.c. 

Allow  time  for  true  solution.  (About  three  days  is 
sufficient.) 

METHOD. — 

1.  Prepare  film,  dry,  but  do  not  fix. 

2.  Flood  the  unfixed  film  with  the  stain,  allow  it  to 
act  for  3  minutes  (the  methylic  alcohol  of  the  stain 
fixes  the  film) . 

3.  Pour  off  the  stain  and  wash  in  distilled  water  until 
the  film  presents  a  pink  colour. 

4.  Dry  and  mount. 

Leishman's  Stain. — 

A.  Weigh  out: 

Methylene  blue  (medicinal)       .    .    .    i  gramme 
Dissolve  in 
Sodium  carbonate,  o .  5  per  cent,  aqueous  solution  .    .    100  c.c. 

Keep  at  65°  C.  for  12  hours  in  either  a  hot  incubator 
or  a  water-bath;  then  stand  in  dark  place  at  room 
temperature  (20°  C.)  for  ten  days. 

B.  Weigh  out : 

Eosin,  extra  B.  A o.i  gramme 

Dissolve  in 

Distilled  water 100  c.c. 

i.  Mix  the  two  solutions  A    and  B    in  equal  volumes, 


CAPSULE    STAINS  99 

and  allow  the  mixture  to  stand  for   12  hours  with 
occasional  stirring. 

2.  Filter,    and   collect   precipitate   on   filter   paper. 

3.  Wash  precipitate  thoroughly  with  distilled  water, 
and  dry. 

4.  Weigh  out  0.15  gramme  of  the  dried  precipitate; 
rub  up  in  a  mortar  with  5    c.c.  of  methylic  alcohol 
(Merck's  puriss,  for  analysis) . 

Allow  undissolved  powder  to  settle,  then  decant  the 
supernatant  fluid  to  a  clean  100  c.c.  measuring  cylinder. 

5.  Add  further  5  c.c.  alcohol  to  sediment  in  mortar 
and  repeat  the  process,  and  so  on  until  all  the  sediment 
has  been  dissolved. 

6.  Now  make  up  the  fluid  in  the  measuring  cylinder 
to  100  c.c.  by  the  addition  of  more  methylic  alcohol. 

METHOD. — 

1.  Prepare  film,  dry,  but  do  not  fix. 

2.  Flood  the  unfixed  film  with  stain,  allow  it  to  act 
30  seconds. 

3.  Add  double  the  volume  of  distilled  water  to  the 
stain  on  the  film,  and  mix  with  glass  rod  or  platinum 
loop. 

4.  Allow  this  diluted  stain  to  act  five  minutes. 

5.  Wash  off  with  distilled  water. 

6.  Leave  some  water  on  film  for  thirty  seconds  to  in- 
tensify the  colour  contrasts. 

7.  Dry  and  mount. 

METHODS  OF  DEMONSTRATING  STRUCTURE  OF 
BACTERIA,  ETC. 

To  Demonstrate  Capsules. 
1.  MacConkey. — 

Stain. — 
Weigh  out 

Dahlia      0.5  gramme 

Methyl  green  (oo  crystals) z  •  5  grammes 


100  STAINING   METHODS 

rub  up  in  a  mortar  with 

Distilled  water loo.oc.c. 

Add 

Fuchsin,  saturated  alcoholic  solution     .    .    .     lo.oc.c. 

and  make  up  to  200  c.c.  by  the  addition  of 

Distilled  water      90.0  c.c. 

Filter. 

Allow  the  stain  to  stand  for  two  weeks  before  use; 
keep  in  a  dark  place  or  in  an  amber  glass  bottle.  Owing 
to  the  unstable  character  of  the  methyl  green,  this 
stain  deteriorates  after  about  six  months. 

METHOD.— 

1.  Prepare  and  fix  film  in  the  usual  manner. 

2.  Flood  the  cover-slip  with  the  stain  and  allow  it 
to  act  for  five  to  ten  minutes. 

3.  Wash   very  thoroughly  in   water;   if   necessary, 
direct  a  powerful  stream  of  water  on  the  film  from  a 
wash-bottle. 

4.  Dry  and  mount. 

2.  Muir's  Method.— 

1.  Prepare,  dry  and  fix  film  in  the  ordinary  manner. 

2.  Flood    the    film    with    carbolic   fuchsin,    warm    until   steam 
begins  to  rise.     Allow  the  stain  to  act  for  thirty  seconds. 

3.  Wash  quickly  with  methylated  spirit. 

4.  Wash  thoroughly  with  water. 

5.  Subject  the  film  to  the  action  of  the  following  mordant  for 
five  seconds: 

Corrosive  sublimate,  saturated  aqueous  solution  .  2  c.c. 
Tannic  acid,  20  per  cent,  aqueous  solution  ...  2  c.c. 
Potash  alum  saturated  aqueous  solution  ....  5  c.c. 

6.  Wash  thoroughly  in  water. 

7.  Treat  with  methylated  spirit  for  about  sixty  seconds.      (The 
preparation  should  now  be  pale  red.) 

8.  Wash  thoroughly  in  water. 

9.  Counterstain   in   methylene   blue,    aqueous    solution    thirty 
seconds. 

10.  Wash  in  water. 

1 1 .  Dehydrate  in  alcohol. 

12.  Clear  in  xylol  and  mount  in  xylol  balsam. 


FLAGELLA    STAINS  IOI 

3.  Welch's  Method.— 

1.  Prepare  and  fix  film  in  the  usual  manner. 

2.  Flood  the  slide  with  acetic  acid  2  per  cent.;  allow  the  acid 
to  remain  in  contact  with  the  film  for  two  minutes.     This  swells 
up  and  fixes  the  capsule  and  enables  it  to  take  the  stain. 

3.  Blow  off  the  acetic  acid  by  the  aid  of  a  pipette. 

4.  Immerse  in  aniline  genotian  violet,  five  to  thirty  seconds. 

5.  Wash  in  water. 

6.  Dry  and  mount. 

4.  Ribbert's  Method.— 

Stain. — 

Measure  out  and  mix: 

Acetic  acid,  glacial I2-5  c-c« 

Alcohol,  absolute 50.0  c.c. 

Distilled  water loo.oc.c. 

Warm  to  36°  C.  (e.  g.,  in  the  "hot"  incubator)  and  saturate 
with  dahlia.  Filter. 

METHOD. — 

1.  Prepare  and  fix  films  in  the  usual  manner. 

2 .  Cover  the  film  with  the  stain  and  allow  it  to  act  for  one  or 
two  seconds  only. 

3.  Wash  thoroughly  in  water. 

4.  Dry  and  mount. 

To  Demonstrate  Flagella. 

1.  Muir's  Modified  Pitfield.— This  is  the  best  method 
and  gives  the  most  reliable  results,  for  not  only  is  the 
percentage  of  successful  preparations  higher  than  with 
any  other,  but  the  bacilli  and  flagella  retain  their  rela- 
tive proportions. 

(a)  Mordant. — 

Tannic  acid,  10  per  cent,  aqueous  solution  .    .    .  10  c.c. 

Corrosive  sublimate,  saturated  aqueous  solution .  5  c.c. 

Alum,  saturated  aqueous  solution 5  c.c. 

Carbolic  fuchsin  (Ziehl) 5  c-c- 

Mix  thoroughly. 

A  precipitate  forms  which  must  be  allowed  to  settle 
for  a  few  hours. 

Decant  off  the  clear  fluid  into  tubes  and  centrifugalise 
thoroughly. 


102  STAINING   METHODS 

This  solution  is  at  its  best  some  four  or  five  days 
after  manufacture ;  it  keeps  for  about  a  couple  of  weeks, 
but  must  be  re-centrifugalised  each  time,  before  use. 

(b)  Stain.— 

Alum,  saturated  aqueous  solution 25  c.c. 

Gentian  violet,  saturated  alcoholic  solution     .    .      5  c.c. 

Filter. 

This  stain  must  be  freshly  prepared. 

METHOD. — The  cultivations  employed  should  be 
smear  agar  cultures,  twelve  to  eighteen  hours  old  if 
incubated  at  37°  C.,  twenty-four  to  thirty  hours  if 
incubated  at  22°  C. 

1.  Remove  a  very  small  quantity  of  the  growth  by 
means  of  the  platinum  spatula. 

2.  Emulsify  it  with  a  few  cubic  centimetres  of  dis- 
tilled water  in  a  watch-glass,  by  gently  moving  the 
spatula  to  and  fro  in  the  water.     Do  not  rub  up  the 
growth  on  the  side  of  the  watch-glass.     Some  workers 
prefer  to  use  tap  water,  others  employ  normal  saline 
solution,  but  distilled  water  gives  the  best  emulsion. 

3.  Spread  a  thin  film  of  the  emulsion  on  a  newly 
flamed  cover-slip,  using  no  force,  but  rather  leading 
the  drop  over  the  cover-slip  with  the  platinum  loop. 

4.  Allow  the  film  to  dry  in  the  air,  properly  protected 
from  falling  dust. 

5.  Fix  by  passing  thrice  through  the  Bunsen  flame, 
holding  the  cover-slip  whilst  doing  so  by  one  corner 
between  the  ringer  and  thumb. 

6.  Pour  on  the  film  as  much  of  the  mordant  as  the 
cover-glass  will  hold.     Grasp  the  cover-slip  with  the 
forceps  and  hold  it,  high  above  the  flame,  until  steam 
rises.     Allow  the  steaming  mordant  to  remain  in  con- 
tact with  the  film  two  minutes. 

7.  Wash  well  in  water  and  dry  carefully. 

8.  Pour  on  the  film  as  much  of  the  stain  as  the  cover- 
glass  will  hold.     Steam  over  the  flame  as  before  for 
two  minutes. 


FLAGELLA    STAINS  103 

9.  Wash  well  in  water. 

10.  Dry  and  mount. 

2.   "Pitfield"  Original  Method.— 

(a)  Mordant. — 

Tannic  acid i  gramme 

Water 10  c.c. 

(b)  Stain.— 

Saturated  aqueous  solution  of  alum 10  c.c. 

Saturated  alcoholic  solution  of  gentian  violet.    .      i  c.c. 
Distilled  water 5  c.c. 

Mix  equal  parts  of  a  and  b  before  using. 

1.  Prepare  and  fix  the  film  in  the  manner  described  above. 

2.  Boil  the  mixture  and  immerse  the  cover-slip  in  it,  whilst 
still  hot,  for  one  minute. 

3.  Wash  in  water. 

4.  Examine  in  water;  if  satisfactory,  dry  and  mount  in  Canada 
balsam. 

3.  MacCrorrie's  Method.— 

Mordant-Stain. — 
Measure  out  and  mix. 

Night  blue,  saturated  alcoholic  solution  .  .10  c.c. 
Potash  alum,  saturated  aqueous  solution  .  10  c.c. 
Tannin,  10  per  cent,  aqueous  solution  .  .10  c.c. 

NOTE. — The  addition  of  gallic  acid,  o.i  to  0.2  gramme,  may 
improve  the  solution,  but  is  not  necessary. 

METHOD. — 

1.  Prepare  and  fix  the  films  as  above. 

2.  Pour   some   of   the   mordant-stain   on   the   film    and    warm 
gently,  high  above  the  flame,  for  two  minutes  (or  place  in  the 
"hot"  incubator  for  a  like  period). 

3.  Wash  thoroughly  in  water. 

4.  Dry  and  mount. 

4.  Loeffler's  Method. — 

(a)   Mordant. — 

Tannic  acid,  20  per  cent,  aqueous  solution      .    .  10  c.c. 

Ferrous  sulphate,  saturated  aqueous  solution      .  5  c.c. 

Haematoxylin  solution 3  c-c- 

Carbolic  acid,  i  per  cent,  aqueous  solution      .    .  4  c.c. 

This  solution  must  be  freshly  prepared. 

Hcematoxylin  solution  is  prepared  by  boiling  i  gramme  logwood 


104  STAINING   METHODS 

with  8  c.c.  distilled  water,  filtering  and  replacing  the  loss  from 
evaporation. 

Alternative  Mordant  (Bunge's  Mordant). — 

Tannic  acid,  20  per  cent,  aqueous  solution  .  .  10  c.c. 
Ferrous  sulphate,  saturated  aqueous  solution .  .  5  c.c. 
Fuchsin,  saturated  alcoholic  solution i  c.c. 

(b)   Stain.— 

Weigh  out 

Methylene-blue ] 

Or  methylene-violet     ....  j>  4  grammes 

Or  fuchsin J 

and  dissolve  in 

Aniline  water,  freshly  saturated  and  filtered  ...  100  c.c. 

METHOD. — 

1.  Prepare  and  fix  films  as  above. 

2.  Pour  the  mordant  on  to  the  film  and  warm  cautiously  over 
the  flame  till  steam  rises;  keep  the  mordant  gently  steaming  for 
one  minute. 

3 .  Wash  well  in  distilled  water  till  no  more  colour  is  discharged ; 
if  necessary,  wash  carefully  with  absolute  alcohol. 

4.  Filter  a  few  drops  of  the   stain  on  to  the  film,   warm   as 
before,  and  allow  the  steaming  stain  to  act  for  one  minute. 

5.  Wash  well  in  distilled  water. 

6.  Dry  and  mount. 

NOTE. — The  flagella  of  some  organisms  can  be  demonstrated 
better  by  means  of  an  alkaline  stain  or  an  acid  stain — a  point  to 
be  determined  for  each.  Speaking  generally,  those  bacilli  which 
give  rise  to  an  acid  reaction  in  the  culture  medium  require  an 
alkali;  those  which  form  alkali  in  cultivation  require  an  acid. 
According  to  requirements,  therefore,  Loeffler  recommends  the 
addition  of  sodium  hydrate,  i  per  cent,  aqueous  solution,  i  c.c.; 
or  an  equal  quantity  of  an  exactly  comparable  solution  of  sul- 
phuric acid. 

5.  Van  Ermengem's  Method. — This  method,  being  merely  a 
precipitation  of  a  silver  salt  on  the  micro-organisms  and  not  a 
true  stain,  creates  a  false  impression  as  to  the  relative  proportions 
of  bacteria  and  flagella. 

(a)  Fixing  Fluid. — 

Osmic  acid,  2  per  cent,  aqueous  solution  .  10  c.c. 
Tannic  acid,  20  per  cent,  aqueous  solution  .  20  c.c. 
Acetic  acid,  glacial i  c.c. 


FLAGELLA    STAINS  105 

The  fixing  fluid  should  be  prepared  some  days  before  use  and 
filtered  as  required.     In  colour  it  should  be  distinctly  violet. 

(6)   Sensitising  Solution. — 

Silver  nitrate,  0.5  per  cent,  aqueous  solution. 

This  solution  must  be  kept  in  a  dark  blue  glass  bottle  or  in  a 
dark  cupboard. 
Filter  immediately  before  use. 

(c)  Reducing  Solution. — 
Weigh  out 

Gallic  acid 5  grammes 

Tannic  acid 3  grammes 

Potassium  acetate,  fused 10  grammes 

and  dissolve  in 

Distilled  water 350  c.c. 

Filter. 

This  solution  will  keep  active  for  several  days,  but  fresh  solution 
must  be  used  for  each  preparation. 

METHOD. — 

1.  Prepare  emulsion,  make  and  fix  films  as  above  in  the  preced- 
ing method,  steps  i  to  4. 

2.  Pour  on  the  film  as  much  of  the  fixing  solution  as  the  cover- 
glass  will  hold,  heat  carefully  over  the  flame  till  steam  rises,  and 
allow  the  steaming  fixing  fluid  to  act  for  five  minutes. 

3.  Wash  well  in  water. 

4.  Wash  in  absolute  alcohol. 

5.  Wash  in  distilled  water. 

6.  Pour  some  of  the  sensitising  solution  on  the  film  and  allow 
it  to  act  for  from  thirty  seconds  to  one  minute ;  blot  off  the  excess 
of  fluid  with  filter  paper. 

7.  Without  washing,  transfer  the  film  to  a  watch-glass  contain- 
ing the  reducing  solution  and  allow  it  to  remain  therein  for  from 
thirty  seconds  to  one  minute;  blot  off  the  excess  of  fluid  with 
filter  paper. 

8.  Without  washing,  again  treat  the  film  with  the  sensitising 
solution,  this  time  until  the  film  commences  to  turn  black. 

9.  Wash  in  distilled  water. 

10.  Dry  and  mount. 

To  Stain  Nuclei  of  Yeast  Cells. 

1.  Prepare  and  fix  film  in  the  usual  manner. 

2.  Soak  in   ferric   ammonia   sulphate    3    per   cent, 
aqueous  solution  for  two  hours. 


106  STAINING   METHODS 

3.  Wash  thoroughly  in  water. 

4.  Stain  in  haematoxylin  solution  (see  page  95)  for 
thirty  minutes. 

5.  Wash  in  water. 

6.  Differentiate  in  ferric  ammonia  sulphate  solu- 
tion for  iJ-2  minutes,  examining   wet   under  micro- 
scope during  the  process. 

To  Stain  Spores. 
1.  Single  Stain. — 

1.  Prepare  cover-slip  film  in  the  usual  way. 

2.  In  fixing,  pass  the  cover-slip  film  fifteen  or  thirty 
times  through  the  flame  instead  of  only  three.     This 
destroys  the  resisting  power  of  the  spore  membrane 
and  allows  the  stain  to  reach  the  interior. 

3.  Stain  in  the  usual  way  with  methylene-blue  or 
fuchsin. 

4.  Wash  in  water. 

5.  Dry  and  mount. 

2.  Double  Stain.— 

1.  Prepare  and  fix  film  in  the  usual  way — i.  e.,  pass 
three  times  through  flame  to  fix. 

2.  Cover  the  film  with  hot  carbol-fuchsin  and  hold 
in  the  forceps  above  a  small  flame  until  the  fluid  begins 
to  steam.     Set  the  cover-slip  down  and  allow  it  to 
cool.     Repeat  the  process  when  the  stain  ceases  to 
steam  and  continue  to  repeat  until  the  stain  has  been 
in  contact  with  the  film  for  twenty  minutes.     (This 
stains  both  spores  and  bacteria.) 

3.  Wash  in  water. 

4.  Decolourise  in  alcohol,  2  parts;  acetic  acid,  i  per 
cent.,  i  part.     (This  removes  the  stain  from  everything 
but  the  spores.) 

5.  Wash  in  water. 

6.  Mount  the  cover-slip  in  water  and  examine  micro- 
scopically with  the  J-inch  objective.     (Spores  should 


SPORE    STAINS  107 

be  red,  and  the  rest  of  the  film  colourless  or  a  very 
light  pink.)  If  satisfactory,  pass  on  to  section  7;  if 
unsatisfactory,  repeat  steps  2  to  5. 

7.  Counterstain    in    weak    methylene-blue.     (Now 
spores  red,  bacilli  blue.) 

8.  Wash  in  water. 

9.  Dry  and  mount. 

The  spores  of  different  bacilli  differ  greatly  in  their 
resistance  to  decolourising  reagents;  even  the  spores 
of  the  same  species  of  organisms  vary  according  to 
their  age.  Young  spores  are  more  easily  decolourised 
than  those  more  mature. 

Sulphuric  acid,  i  per  cent,  aqueous  solution,  and 
hydrochloric  acid,  0.5  per  cent,  alcoholic  (90  per  cent.) 
solution,  are  useful  decolourising  reagents. 

3.  Moeller's  Method.— 

1.  Prepare  and  fix  films  in  the  usual  manner. 

2.  Immerse    in    absolute    alcohol    for    two    minutes,    then    in 
chloroform  for  two  minutes;  wash  in  water.     This  dissolves  out 
any  fat  or  Crystals  that  might  otherwise   retain   the   "spore" 
stain. 

3.  Immerse  in  chromic  acid,  5  per  cent,  aqueous  solution,  for 
one  minute;  wash  in  water. 

4.  Pour  Ziehl's  carbolic  fuchsin  on  the  film,  warm  as  in  previous 
methods,  and  allow  it  to  act  for  ten  minutes. 

5.  Wash  in  water. 

6.  Decolourise  in  sulphuric  acid,  5  per  cent,  aqueous  solution, 
for  five  seconds. 

7.  Wash  in  water. 

8.  Counterstain  with  Kuehne's  carbolic  methylene-blue  for  one 
or  two  minutes. 

9.  Wash  in  water. 

10.  Dry  and  mount. 
(Spores  red,  bacilli  blue.) 

4.  Abbott's  Method.— 

1.  Prepare  and  fix  films  in  the  usual  manner. 

2.  Pour  Loeffler's  alkaline  methylene-blue  on  the  film;  warm 
cautiously   over   the  flame  till   steam   rises   and   allow    the    hot 
steam  to  act  for  one  to  five  minutes. 

3.  Wash  thoroughly  in  water. 

4.  Decolourise  in  nitric  acid,    2    per  cent,    alcoholic    (alcohol 
80    per  cent.)  solution. 


108  STAINING   METHODS 

5.  Wash  thoroughly  in  water. 

6.  Counterstain  in  eosin,  i  per  cent,  aqueous  solution. 

7.  Wash. 

8.  Dry  and  mount. 
(Spores  blue,  bacilli  red.) 

DIFFERENTIAL  METHODS  OF  STAINING. 

Gram's  Method. — This  method  depends  upon  the 
fact  that  the  protoplasm  of  some  bacteria  permits 
aniline  gentian  violet  and  Lugol's  iodine  solution,  when 
applied  consecutively,  to  enter  into  a  chemical  combina- 
tion which  results  in  the  formation  of  a  new  blue-black 
pigment,  only  very  sparingly  soluble  in  absolute 
alcohol.  Such  organisms  are  said  to  "  stain  by  Gram,'* 
or  to  be  "Gram  positive." 

1.  Prepare  a  cover-slip  film  and  fix  in  the  usual  way. 

2 .  Stain  in  aniline  gentian  violet  three  to  five  minutes. 
Filter  as  much  aniline  water  on  to  the  cover-slip  as 

it  will  hold ;  then  add  the  smallest  quantity  of  alcoholic 
solution  of  gentian  violet  which  suffices  to  saturate  the 
aniline  water  and  form  a  "bronze  scum"  upon  its 
surface  — if  too  much  of  the  alcoholic  gentian  violet  is 
added  the  alcohol  present  redissolves  this  scum. 

To  prepare  aniline  water,  pour  4  or  5  c.c.  aniline  oil  into  a 
stoppered  bottle  and  add  distilled  water,  100  c.c.  Shake  vigour- 
ously  and  filter  immediately  before  use.  The  excess  of  oil  sinks 
to  the  bottom  of  the  bottle  and  may  be  used  again. 

3.  Wash  in  water. 

4.  Treat  with  Lugol's  iodine  solution  until  the  film 
is  black  or  dark  brown. 

To  do  this  treat  with  iodine  solution  for  a  few  seconds, 
wash  in  water,  and  examine  the  film  over  a  piece  of 
white  filter  paper.  Note  the  colour.  Repeat  this  proc- 
ess until  the  film  ceases  to  darken  with  the  fresh  appli- 
cation of  iodine  solution. 

Lugol's  solution  is  prepared  by  dissolving 

Iodine       i  gramme 

Iodide  of  potassium 3  grammes 

In  distilled  water 300  c.c. 


DIFFERENTIAL   STAINS  109 

5.  Wash  in  water. 

6.  Wash  with  alcohol  until  no  more  colour  is  dis- 
charged and  the  alcohol  runs  away  clear  and  colourless. 

The  following  mixture  may  be  substituted  for  abso- 
lute alcohol  as  a  decolouriser 

Acetone 10  c.c. 

Absolute  alcohol      100  c.c. 

7.  Wash  in  water. 

8.  Counterstain  very  lightly  with  aqueous  solution  of 
Neutral  Red.     Other  counterstains  may  be  used  such 
as  dilute  eosin,  dilute  fuchsin,  or  vesuvin. 

NOTE. — This  section  may  be  omitted  when  dealing  with  films 
prepared  from  pure  cultivations. 

9.  Wash  in  water. 

10.  Dry  and  mount. 

Gram=Claudius  Method.— 

1 .  Prepare  a  cover-slip  film  and  fix  in  the  usual  way. 

2.  Stain  in  methyl  violet,  i  per  cent,  aqueous  solution 
for  three  to  five  minutes. 

3.  Treat  with  two  lots  picric  acid,  saturated  aqueous 
solution. 

4.  Wash  in  water  and  dry. 

5.  Decolourise  with  clove  oil. 

6.  Wash  off  clove  oil  with  xylol. 

7.  Mount  in  xylol  balsam. 

Qram=Weigert  Method. — 

1-5.  Proceed  as  for  the  corresponding  sections  of 
Gram's  method  (quod  vide) . 

6.  Dry  in  the  air. 

7.  Wash  in  aniline  oil,  i  part,  xylol,  2  parts,  until 
no  more  colour  is  discharged. 

8.  Wash  in  xylol. 

9.  Mount  in  xylol  balsam. 


110  STAINING   METHODS 

Modified  Gram=Weigert  Method. — (To  demonstrate 
trichophytain  hair.) 

1.  Soak  the  hairs  in  ether  for  ten  minutes  to  remove 
the  fat. 

2.  Stain   thirty   minutes  in   a  tar-like   solution   of 
aniline  gentian  violet  (prepared  by  adding  1 5  drops  of 
the  alcoholic  solution  of  gentian  violet  to  3  drops  of 
aniline  water) . 

3.  Dry  the  hairs  between  pieces  of  blotting  paper. 

4.  Treat  with  perfectly  fresh  iodine  solution. 

5.  Again  dry  between  blotting  paper. 

6.  Treat  with  aniline  oil  to  remove  excess  of  stain. 
(If  necessary,  add  a  drop  or  two  of  nitric  acid  to  the  oil.) 

7.  Again  treat  with  aniline  oil. 

8.  Treat   with  aniline  oil  and   xylol,   equal  parts. 

9.  Clear  with  xylol. 

10.  Mount  in  xylol  balsam. 

To  obtain  the  best  differentiation  the  preparation 
should  be  repeatedly  examined  microscopically  (with 
a  J-inch  objective)  between  steps  5  and  9,  as  the  actual 
time  involved  varies  with  different  specimens. 

Ziehl=Neelsen's  Method. — (To  demonstrate  tubercle 
and  other  acid-fast  bacilli.) 

1.  Smear  a  thin,  even  film  of  the  specimen  on  the 
cover-slip  by  means  of  the  platinum  loop.     (In  the  case 
of  sputum,  if  it  is  a  very  watery  specimen,  allow  the 
film  to  dry,  then  spread  a  second  and  even  a  third  layer 
over  the  first.) 

2.  Fix  by  passing  three  times  through  the  flame. 

3.  Stain  in  hot  carbol-fuchsin   (as  in  staining  for 
spores)  for  five  to  ten  minutes.     (This  stains  every- 
thing on  the  film.)     Avoid  over-heating. 

4.  Decolourise  by  dipping  in  sulphuric  acid,  25  per 
cent.     (This  removes  stain  from  everything  but  acid- 
fast  bacilli;  e.  g.,  tubercle,  leprosy,  and  smegma  bacilli 
and  the  film  turns  yellow.) 


DIFFERENTIAL    STAINS  III 

5.  Wash   in   water.     (A  pale  red  colour  returns  to 
the  film). 

6.  Wash  in  alcohol  till  no  more  colour  is  discharged. 
(This    often,    but   not  invariably,   removes  the  stain 
from  acid-fast  bacilli  other  than  tubercle;  e.  g.,  smegma 
bacillus.) 

7.  Wash  in  water. 

8.  Counterstain   in    weak   methylene-blue.     (Stains 
non-acid-fast  bacilli,  leucocytes,  epithelial  cells,  etc.) 

9.  Wash  in  water,  dry,  and  mount. 
Pappenheim's  Method. — 

This  method  is  supposed  to  differentiate   between 
B.  tuberculosis   and  other  acid-fast    micro-organisms. 

1.  Prepare  and  fix  film  in  the  usual  way. 

2.  Stain    in   carbol-fuchsin    without    heat    for    three 
minutes. 

3.  Without  previously  washing  in  water  treat  the 
film  with  three  or  four  successive  applications  of  coralin 
(Rosolic  acid)  solution. 

Corallin i  gramme 

Methylene-blue 

(saturated  alcoholic  solution)  .  100  c.c. 

Glycerine 20  c.c. 

4.  Wash  in  water. 

5.  Dry  and  mount. 

Neisser's  Method— Modified.— (To  demonstrate  diph- 
theroid  bacilli.) 
Stain  I. — 
Measure  out  and  mix 

Methylene-blue,  saturated  alcoholic 

solution 4-0  c.c. 

Acetic  acid,  5  per  cent,  aqueous  solu- 
tion   96.0  c.c. 

Filter. 
Stain  II. — 
Weigh  out 

Neutral  red  2.5  grammes 


112  STAINING   METHODS 

and  dissolve  in 

Distilled  water 1000  c.c. 

Filter. 
METHOD. — 

1.  Prepare  and  fix  films  in  the  usual  way. 

2.  Pour  stain  I  on  the  film  and  allow  it  to  act  for 
two  minutes. 

3.  Wash  thoroughly  in  water. 

4.  Treat  with  Lugol's  iodine  for  ten  seconds. 

5.  Wash  thoroughly  in  water. 

6.  Pour  stain  II  on  to  the  film  and  allow  it  to  act  for 
thirty  seconds. 

7.  Wash  thoroughly  in  water. 

8.  Dry  and  mount. 

NOTE. — The  cultivation  from  which  the  films  are  prepared 
must  be  upon  blood-serum  which  has  been  incubated  at  37°  C. 
for  from  nine  to  eighteen  hours. 

The  bacilli  are  stained  a  light  red  by  the  neutral  red, 
which  contrasts  well  with  the  two  or  three  black  spots, 
situated  at  the  poles  and  occasionally  one  in  the  centre 
representing  protoplasmic  aggregations  (?  meta-chro- 
matic  granules)  stained  by  the  acid  methylene-blue. 

Wheal  and  Chown  (Oxford)  Method.— (To  demonstrate  actino- 
myces.) 

1.  Stain   briefly  with   Ehrlich's   haematoxylin  (until  nuclei  are 
faint  blue  after  washing  with  tap  water) . 

2.  Wash  in  tap  water. 

3.  Stain  in  hot  carbol-fuchsin  (as  for  tubercle  bacilli)  for  five 
to  ten  minutes. 

4.  Wash  in  tap  water. 

5.  Decolourise    with    Spengler's    picric    acid    alcohol.     This    is 
prepared  by  mixing: 

Alcohol,  absolute 20  c.c. 

Picric  acid,  saturated  aqueous  solution    10  c.c. 
Distilled  water 10  c.c. 

During  the  progress  of  steps  1-5  the  preparation  must  be  re- 
peatedly examined  microscopically  with  the  ^-inch  objective. 


DIFFERENTIAL    STAINS  113 

When  properly  differentiated  the  clubs  appear  brilliant  red  on 
greenish  ground. 

6.  Dehydrate  in  alcohol. 

7.  Clear  in  xylol. 

8.  Mount  in  xylol  balsam. 

This  method  serves  equally  well  for  films  and  for  sections. 


VII.  METHODS  OF  DEMONSTRATING  BACTERIA 
IN  TISSUES. 

FOR  bacteriological  purposes,  sections  of  tissue  are 
most  conveniently  prepared  by  either  the  freezing 
method  or  the  paraffin  method. 

The  latter  is  decidedly  preferable,  but  as  it  is  of 
greater  importance  to  demonstrate  the  bacteria,  if  such 
are  present,  than  to  preserve  the  tissue  elements  un- 
altered, the  "frozen"  sections  are  often  of  value. 

Whichever  method  is  selected,  it  is  necessary  to  take 
small  pieces  of  the  tissue  for  sectioning, — 2  to  5  mm. 
cubes  when  possible,  but  in  any  case  not  exceeding  half 
a  centimetre  in  thickness.  Post-mortem  material  should 
be  secured  as  soon  after  the  death  of  the  animal  as 
possible. 

The  tissue  is  prepared  for  cutting  by— 

(a)  Fixation;  that  is,  by  causing  the  death  of  the 
cellular  elements  in  such  a  manner  that  they  retain 
their  characteristic  shape  and  form. 

The  fixing  fluids  in  general  use  are :  Absolute  alcohol ; 
corrosive  sublimate,  saturated  aqueous  solution;  cor- 
rosive sublimate,  Lang's  solution  (vide  page  82) ; 
formaldehyde,  4  per  cent,  aqueous  solution.  (Of 
these,  Lang's  corrosive  sublimate  solution  is  decidedly 
the  best  all-round  " fixative.") 

(6)  Hardening;  that  is,  by  rendering  the  tissue  of 
sufficient  consistency  to  admit  of  thin  slices  or  "  sec- 
tions" being  cut  from  it.  This  is  effected  by  passing 
the  tissue  successively  through  alcohols  of  gradually 
increasing  strength:  30  per  cent,  alcohol,  50  per  cent, 
alcohol,  75  per  cent,  alcohol,  90  per  cent*,  alcohol, 
absolute  alcohol. 

In  both  these  processes  a  large  excess  of  fluid  should 
always  be  used. 

114 


HARDENING 
FREEZING  METHOD. 

1.  Fixation.     Place  the  pieces  of  tissue  in  a  wide- 
mouthed  glass  bottle  and  fill  with  absolute  alcohol.     Al- 
low the  tissues  to  remain  therein  for  twenty-four  hours. 

2.  Hardening.     Remove  the  alcohol  (no  longer  abso- 
lute, as  it  has  taken  up  water  from  the  tissues)  from 


FIG.  71. — Washing  tissues. 

the  bottle  and  replace  it  with  fresh  absolute  alcohol. 
Allow  the  tissues  to  remain  therein  for  twenty-four 
hours. 

NOTE. — If  not  needed  for  cutting  immediately,  the  hardened 
tissues  can  be  stored  in  75  per  cent,  alcohol. 

3.  Remove  the  alcohol  from  the  tissues  by  soaking  in 
water  from  one  to  two  hours.  Remove  the  stopper 
from  the  bottle;  rest  a  glass  funnel  in  the  open  mouth 


Il6  DEMONSTRATING    BACTERIA   IN    TISSUES 

and  place  under  a  tap  of  running  water.  The  water  of 
course,  overflows,  but  the  tissues  remain  in  the  bottle 
(Fig.  71). 

4.  Impregnate  the  tissues  with  mucilage  for  twelve 
to  twenty-four  hours,  according  to  size.     Transfer  the 
pieces  of  tissue  to  a  bottle  containing  sterilised  gum 
mixture. 

Formula.— 

Gum  arable 5  grammes 

Saccharose i  gramme 

Boric  acid i  gramme 

Water 100  c.c. 

5.  Place  the  tissue  on  the  plate  of  a  freezing  micro- 
tome (Cathcart's  is  perhaps  the  best  form),  cover  and 
surround  with  fresh  gum  mixture ;  freeze  with  ether,  or 
for    preference,    carbon    dioxide,    and    cut    sections. 

6.  Float  the  sections  off  the  knife  into  a  glass  dish 
containing   tepid   water   and   allow   them   to   remain 
therein  for  about  an  hour  to  dissolve  out  the  gum. 

(If  not  required  at  once,  store  in  90  per  cent,  alcohol.) 

7.  Transfer  to  a  glass  capsule  containing  the  selected 
staining  fluid,  by  means  of  a  section  lifter. 

8.  Transfer  the  sections  in  turn  to  a  capsule  con- 
taining absolute  alcohol   (to  dehydrate)   and   to   one 
containing  xylol  or  oil  of  cloves  (to  clear). 

9.  Mount  in  xylol  balsam. 

Alternative  Rapid  Method. — 

1.  Cut  very  small  blocks  of  the  tissue. 

2.  Fix  in  formalin  10  per  cent,  aqueous  solution  (fixation  fluid 
No.  7,  page  82)  for  24  hours. 

3.  Transfer  block  to  plate  of  freezing  microtome  and  freeze  with 
carbon  dioxide  vapour. 

4.  Float  the  sections  off  the  knife  into  a  glass  dish  of  tepid 
water. 

5.  Stain  the  sections  in  glass  capsules  containing  selected  stains. 

6.  Place    the    stained    section  in   a  dish  of  clean  water  and 
introduce   a  glass   slide  obliquely  beneath  the   section;   with   a 
mounted  needle  draw  the  section  on  to  the  slide  and  hold  it  there; 


CLEARING 


117 


gently  remove  the  slide  from  the  water,  taking  care  that  any 
folds  in  the  section  are  floated  out  before  the  slide  is  finally 
removed  from  the  water. 

7.  Drain    away  as   much  water  as   possible  from  the  section. 
Drop  absolute  alcohol  on  to  the  section  from  a  drop  bottle,  to 
dehydrate  it. 

8.  Double  a  piece  of  blotting  paper  and  gently  press  it  on  the 
section  to  dry  it. 

9.  Drop  on  xylol  to  clear  the  section. 

10.  Place  a  large  drop  of  xylol  balsam  on  the  section  and  care- 
fully lower  a  cover-glass  on  to  the  balsam. 

PARAFFIN  METHOD. 

1.  Fixation.     Place  the  pieces  of  tissue,  resting  on 
cotton- wool,  in  a  wide-mouthed  glass  bottle.     Pour  on 
a  sufficient  quantity  of  the  corrosive  sublimate  fixing 
fluid;  allow  the  tissue  to  remain  therein  for  twelve  to 
twenty-four  hours  according  to  size. 

2.  Pour  off  the  fixing  fluid  and  wash  thoroughly  in 
running  water  for  twenty  minutes  to  half  an  hour  to 
remove  the  excess  of  corrosive  sublimate. 


FIG.  72. — L-shaped  brass  moulds. 


FIG.  73. — Paraffin  kettle. 


3.  Hardening.     Place  the  tissues  in  each  of  the  fol- 
lowing strengths  of  alcohol  in  turn  for  from  twelve  to 
twenty-four  hours:  50  per  cent.,  75  per  cent.,  90  per 
cent.,  absolute. 

4.  Dehydration  is  effected  by  transferring  the  tissues 
to  fresh  absolute  alcohol. 

5.  Clearing.     Half  fill  a  wide-mouthed   bottle  with 


Il8  DEMONSTRATING    BACTERIA   IN    TISSUES 

chloroform.  On  the  surface  of  the  chloroform  float 
a  layer  of  absolute  alcohol  about  five  to  ten  millimetres 
in  depth.  Place  the  pieces  of  tissue  in  the  layer  of 
alcohol  and  when  they  have  sunk  through  this  layer, 
transfer  them  to  pure  chloroform  for  from  six  to 
twenty-four  hours  according  to  the  size  of  the  pieces. 
When  "cleared,"  the  tissue  becomes  more  or  less 
transparent. 

6.  Infiltration.    Place  the  cleared  tissues  in   fresh 
chloroform  with  several   pieces  of   paraffin  wax  and 
stand  in  a  warm  place,  such  as  on  the  top  of  the  warm 
incubator.     The  warmth  gradually  melts  the  paraffin 
and  the  tissues  should  remain  in  the  mixture  about 
twenty-four  hours. 

7.  Transfer  the  tissues  to  a  vessel  containing  pure 
melted  paraffin.     Place  this  vessel  in  a  paraffin  water- 
bath  regulated  for  2°  C.  above  the  melting-point  of  the 
paraffin  used,  and  allow  the  tissues  to  soak  for  some 
four  to  six  hours  to  ensure  complete  impregnation. 
The  paraffin  used  should  have  a  melting-point  of  not 
more  than  58°  C.     For  all  ordinary  purposes  54°  C. 
will  be  found  quite  high  enough. 

8.  Imbed  in  fresh  paraffin  in  a  metal   (or  paper) 
mould. 

(a)  Arrange  a  pair  of  L-shaped  pieces  of  metal  on 
a  plate  of  glass  to  form  a  rectangular  trough  (Fig.  72). 

(b)  Pour  fresh  melted  paraffin  into  the  mould  from  a 
special  vessel  (Fig.  73). 

(c)  Lift  the  piece  of  tissue  from  the  paraffin  bath 
and  arrange  it  in  the  mould. 

(d)  Blow  gently  on  the  surface  of  the  paraffin  in  the 
mould,  and  as  soon  as  a  film  of  solid  paraffin  has  formed, 
carefully  lift  the  glass  plate  on  which  the  mould  is  set 
and  lower  plate  and  mould  together  into  a  basin  of 
cold  water. 

(e)  When  the  block  is  cold,  break  off  the  metal  L's ; 
trim  off  the  excess  of  paraffin  from  around  the  tissue 


MOUNTING    PARAFFIN    SECTIONS  1 19 

with  a  knife,   taking  care  to  retain  the  rectangular 
shape,  and  store  the  block  in  a  pill-box. 

When  several  pieces  of  tissue  have  to  be  imbedded  at 
one  time,  shapes  of  stout  copper,  10  cm.,  5  cm.,  and 
2.5  cm.  square  respectively,  and  0.75  cm.  deep  (Fig.  74) 
will     be     found     extremely     useful. 
These   placed   upon    plates    of  glass 
replace  the  pair  of  L's  in  the  above 
process.     When  the  paraffin  has  set 
firmly  the  screw  a  should  be  loosened 
to  allow  the  two  halves  of  the  flange  b 
to  separate  slightly — this  facilitates    FIG.  74.— Paraffin 
removal  of  the  paraffin  block, 

8.  Cement  the  block  on  the  carrier  of  a  "paraffin" 
microtome    (the  Minot,   the  Jung,   or  the  Cambridge 
Rocker)  with  a  little  melted  paraffin.     Greater  security 
is  obtained  if  the  paraffin  around  the  base  of  the  block  is 
melted  by  means  of  a  hot  metal  or  glass  rod. 

9.  Cut  sections — thin,  and  if  possible  in  ribbands. 

Mounting  Paraffin  Sections. — 

1.  Place  a  large  drop  of  30  per  cent,  alcohol  on  the 
centre  of  a  slide  (or  cover-slip)  and  float  the  section 
on  to  the  surface  of  the  drop,  from  a  section  lifter. 

2.  Hold  the  slide  in  the  fingers  of  one  hand  and  warm 
cautiously  over  the  flame  of  a  Bunsen  burner,  touching 
the  under  surface  of  the  glass  from  time  to  time  on  the 
back  of  the  other  hand.     As  soon  as  the  slide  feels 
distinctly  warm  to  the  skin,  the  paraffin  section  will 
flatten  out  and  all  wrinkles  disappear. 

(The  slide  with  the  section  floating  on  it  may  be 
rested  on  the  top  of  the  paraffin  bath  for  two  or  three 
minutes,  instead  of  warming  over  the  flame  as  here 
described.) 

3.  Cautiously  tilt  up  the  slide  and  blot  off  the  excess 
of    spirit    with    blotting   paper,    leaving    the    section 
attached  to  the  centre  of  the  slide. 


120 


DEMONSTRATING   BACTERIA   IN    TISSUES 


4.  Place  the  slide  in  a  wire  rack  (Fig.  75),  section 
downward,  in  the  "hot"  incubator  for  twelve  to 
twenty-four  hours.  At  the  end  of  this  time  the  section 
is  firmly  adherent  to  the  glass,  and  is  treated  during 
the  subsequent  steps  as  a  "fixed"  cover-glass  film 
preparation. 

NOTE. — If  large,  thick  sections  have  to  be  manipulated,  or  if 
time  is  of  importance  or  acids  are  used  during  the  staining 
process,  it  is  often  advisable  to  add  a  trace  of  Mayer's  albumin 
to  the  alcohol  before  floating  out  the  section.  If  this  substance 
is  employed,  a  sojourn  of  twenty  minutes  to  half  an  hour  in  the 
"hot"  incubator  will  be  found  ample  to  ensure  firm  adhesion 
of  the  section  to  the  slide.  The  albuminous  fluid  is  prepared 
as  follows: 


FIG.  75. — Section  rack. 

Mayer's  Albumin. — 

Weigh  out 

Salicylate  of  soda i  gramme 

and  dissolve  in 

Glycerine 50  c.c. 

Add 

White  of  egg 50  c.c. 

Mix  thoroughly  by  means  of  an  egg  whisk. 

Filter  into  a  clean  bottle. 

As  an  alternative  method  paint  a  thin  layer  of  Schallibaum's 
solution  on  the  slide  with  a  camel's  hair  pencil;  lay  the  section 
carefully  on  this  film  and  heat  gently  to  fix  the  section. 


DOUBLE    STAINING  121 

Schallibaum's  solution: 

Clove  oil 30  c.c. 

Collodion 10  c.c. 

Keep  in  a  dark  blue  bottle  in  a  cool  place. 
Staining  Paraffin  Sections. — 

1.  Warm  paraffin  section  over  the  Bunsen  flame  to 
soften  (but  not  to  melt)  the  paraffin,  then  dissolve  out 
the  wax  with  xylol  poured  on  from  a  drop  bottle. 

2.  Remove  xylol  by  flushing  the  section  with  alcohol. 

3.  If  the  tissue  was  originally  "fixed"  in  a  corrosive 
sublimate  solution,  the  section  must  now  be  treated 
with  Lugol's  iodine  solution  for  two  minutes  and  sub- 
sequently immersed  in  90  per  cent,  alcohol  to  remove 
all  traces  of  yellow  staining. 

4.  Wash  in  water. 

5.  Stain  deeply,  if  using  a  single  stain,  as  the  subse- 
quent processes  decolourise. 

6.  Wash  in  water,  decolourise  if  necessary. 

7.  Flood  with  several  changes  of  absolute  alcohol  to 
dehydrate  the  section. 

8.  Clear  in  xylol.     (Oil  of  cloves  is  not  usually  em- 
ployed, as  it  decolourises  the  section.) 

9.  Mount  in  xylol  balsam. 

SPECIAL  STAINING  METHODS  FOR  SECTIONS. 
Double=staining  Carmine  and  Gram=Weigert. — 

1.  Prepare  the  section  for  staining  as  above,  sections 
i  to  3. 

2.  Stain  in  lithium  carmine  (Orth's)  or  picrocarmine 
for  ten  to  thirty  minutes,  in  a  porcelain  staining  pot 
(Fig.  76). 

3.  Wash  in  picric  acid   solution  until   yellow.     At 
this  stage  cell  nuclei  are  red,  protoplasm  is  yellow,  and 
bacteria  are  colourless. 

Picric  acid  solution  is  prepared  by  mixing 

Picric  acid,  saturated  aqueous  solution       .      40  c.c. 

Hydrochloric  acid       i  c.c. 

Alcohol  (90  per  cent.) 160  c.c. 


122  DEMONSTRATING    BACTERIA   IN    TISSUES 

4.  Wash  in  water. 

5.  Wash  in  alcohol. 

6.  Stain  in  aniline  gentian  violet. 

7.  Wash  in  iodine  solution  till  dark  brown  or  black. 

8.  Wash  in  water. 

9.  Dip  in  absolute  alcohol  for  a  second. 

10.  Decolourise  with  aniline  oil  till  no  more  colour 
is  discharged. 


FIG.  76. — Staining  pot. 

11.  Wash  with  aniline  oil,   2  parts,  xylol,   i  part. 

12.  Clear  with  xylol. 

13.  Mount  in  xylol  balsam. 

Alternative   Qram=Weigert   Method   for   Sections. — 

1.  Fix  paraffin  section  on  slide  and  prepare  for  stain- 
ing in  the  usual  manner. 

2.  Stain  in  alum  carmine  for  about  fifteen  minutes. 

3 .  Wash  thoroughly  in  water. 

4.  Filter  aniline  gentian  violet  solution  on   to  the 
section  on  the  slide  and  allow  to  stain  about  twenty- 
five  minutes. 

5.  Wash  thoroughly  in  water. 

6.  Treat  with  Lugol's  iodine  until  section  ceases  to 
become  any  blacker. 

7.  Wash  thoroughly  in  water. 

8.  Treat  with  a  mixture  of  equal  parts  of  aniline 
oil  and  xylol  until  no  more  colour  comes  away. 


TO   DEMONSTRATE    CAPSULES  123 

9.  Wash  thoroughly  with  xylol. 

10.  Decolourise  and  dehydrate  rapidly  with  abso- 
lute alcohol  until  there  remains  only  a  very  faint  bluish 
tint. 

11.  Clear  with  xylol. 

12.  Mount  in  xylol  balsam. 

(Then  fibrin  and  hyaline  tissue  are  stained  deep  blue, 
whilst  bacteria  which  " stain  Gram"  appear  of  a  deep 
blue- violet  colour.) 

Unna=Pappenheim  Method. — 

Stain. — 
Weigh  out  and  mix 

Methylene  green  . 0.15  gramme 

Pyronin      0.25  gramme 

and  dissolve  in 

Carbolic  acid  0.5  per  cent,  aqueous  solution    .    .    78  c.c. 

Measure  out 

Alcohol 2.5  c.c. 


„.         .  f    and  add  to  the  stain. 

Glycerine 20.0  c.c. 

Method.— 

1.  Place  tissue  in  the  above  stain  for  ten  minutes. 

2 .  Differentiate  and  dehydrate  with  absolute  alcohol. 

3.  Clear  in  xylol. 

4.  Mount  in  xylol  balsam. 

To  Demonstrate  Capsules.— 

1.  MacConkey's    Method. — Stain    precisely    as    for 
cover-slip  films  (vide  page  100). 

2.  Friedldnder's  Method. — 

Stain. — 

Gentian  violet,  saturated  alcoholic  solution       .  50  c.c. 

Acetic  acid,  glacial 10  c.c. 

Distilled  water    .  100  c.c. 


124  DEMONSTRATING   BACTERIA   IN   TISSUES 

METHOD. — 

1.  Prepare  the  sections  for  staining,  secundum  artem. 

2.  Stain  sections  in  the  warm  (e.  g.,  in  the  hot  incubator)  for 
twenty-four  hours. 

3.  Wash  with  water. 

4.  Decolourise  lightly  with  acetic  acid,  i  per  cent. 

5.  Dehydrate  rapidly  with  absolute  alcohol. 

6.  Clear  with  xylol. 

7.  Mount  in  xylol  balsam. 

To  Demonstrate  Acid=fast  Bacilli. — 

1.  Prepare  the  sections  for  staining  in  the  usual  way. 

2.  Stain  with  haematin  solution  ten  to  twenty  sec- 
onds, to  obtain  a  pure  nuclear  stain ;  then  wash  in  water. 

3.  Stain    with    carbolic    fuchsin    twenty    to    thirty 
minutes  at  47°  C. ;  then  wash  in  water. 

4.  Treat   with   aniline   hydrochlorate,    2    per   cent, 
aqueous  solution,  for  two  to  five  seconds. 

5.  Decolourise  in   75  per  cent,  alcohol  till  section 
appears  free  from  stain — fifteen  to  thirty  minutes. 

6.  Dehydrate  with  absolute  alcohol. 

7.  Clear  very  rapidly  with  xylol. 

8.  Mount  in  xylol  balsam. 

To  Demonstrate  Spirochaetes  in  Tissues. 
Piridin  Method  (Levaditi).— 

1.  Cut  slices  of  tissue  i  mm.  thick. 

2.  Fix  in  10  per  cent,  formalin  solution  for  twenty- 
four  hours. 

3.  Wash  in  water  for  one  hour. 

4.  Place  in  96  per  cent,  alcohol  for  twenty-four  hours. 

5.  Measure  into  a  dark  green  or  amber  bottle  100  c.c. 
silver  nitrate  solution  i  per  cent.,  and  10  grammes  pyri- 
din  puriss.     Transfer  slices  of  tissue  to  this.     Stopper 
and  keep  at  room  temperature  three  hours,  then  in 
thermostat  at  50°  C.  for  four  to  six  hours. 

6.  Wash  quickly  in  10  per  cent,  pyridin  solution. 

7.  Reduce  silver  by  transferring  slices  of  tissue  to 
following  solution  for  forty-eight  hours. 


TO    STAIN    PROTOZOA    IN    SECTIONS  125 

Pyrogallic  acid 4  grammes 

Acetone 10  c.c. 

Pyridin  puriss 15  grammes 

Distilled  water 100  c.c. 

8.  Wash  well  in  water. 

Take  through  alcohols  of  increasing  strength  up  to 
absolute,  keeping  in  each  strength  for  twenty-four 
hours. 

9.  Clear,    embed,    cut    very   thin   sections,    mount, 
remove  paraffin,  again  clear  and  mount  in  xylol  balsam. 

The  spirochaetes  if  present  are  black  and  show  up 
against  the  pale  yellow  color  of  the  background. 

Weak  carbol  fuchsin,  neutral  red  or  toluidin  blue 
can  also  be  used  to  stain  the  background  if  desired, 
after  the  removal  of  the  paraffin  in  step  9. 

To  Demonstrate  Protozoa  in  Sections  (Leishman)  .— 

Reagents  required : 

Leishman' s  Polychrome  stain. 
Acetic  acid  i  in  1500  aqueous  solution. 
Caustic  soda  i  in  7000  aqueous  solution. 
Distilled  water. 

1.  Mount  section,   remove    paraffin  and  take  into 
distilled  water  as  usual  (vide  page  121). 

2.  Drain  off  the  excess  of  water. 

3.  Cover  the  section  with  diluted  Leishman  (i  part 
stain,  2  parts  distilled  water)  and  allow  to  act  for  five 
to  ten  minutes  (until  tissue  appears  a  deep  blue) . 

4.  Decolourise  with  acetic  acid  solution  until  only 
the  nuclei  appear  blue  (examine  the  section  wet,  with 
low  power  objective) . 

5.  If  the  eosin  colour  is  too  well  marked  treat  with 
the  caustic  soda  solution  until  the  desired  tint  is  ob- 
tained (as  seen  with  the  J-inch  objective). 

6.  Wash  with  distilled  water. 

7.  Rapidly  dehydrate  with  alcohol. 

8.  Clear  with  xylol. 

9.  Mount  in  xylol  balsam. 


VIII.  CLASSIFICATION  OF  FUNGI. 

For  practical  purposes  FUNGI  may  be  divided  into : 

1.  Hymenomycetes  (including  the  mushrooms,  etc.). 

2.  Hyphomycetes  (moulds). 

3.  Blastomycetes  (yeasts  and  torulae) . 

4.  Schizomycetes  (bacteria). 

NOTE. — Formerly  myxomycetes  were  included  in  the  fungi; 
they  are  now  recognized  as  belonging  to  the  animal  kingdom, 
and  are  termed  "mycetozoa." 

MORPHOLOGY  OF  THE  HYPHOMYCETES. 

At  the  commencement  of  his  studies,  the  attention 
of  the  student  is  directed  to  the  various  non-pathogenic 
moulds  and  yeasts,  not  only  that  he  may  gain  the 
necessary  technique  whilst  handling  cultivations  of 
harmless  organisms,  but  also  because  these  very  species 
are  amongst  the  commonest  of  those  that  may  acci- 
dentally contaminate  his  future  preparations. 

The  hyphomycetes  are  composed  of  a  mycelium  of 
short  jointed  rods  or  "hyphae"  springing  from  an  axis 
or  germinal  tube  which  develops  from  the  spore. 
Hyphae  are — 

(a)  Nutritive  or  submerged. 

(b)  Reproductive  or  aerial. 

The  protoplasm  of  these  cells  contains  granules, 
pigment,  oil  globules,  and  sometimes  crystals  of  cal- 
cium oxalate. 

Reproduction. — Apical     spore    formation — asexual ; 

zoospores — sexual . 

Mucorinae. — Mucor  (Fig.  77). — Note  the  branching 
filaments — "mycelium"  (a),  "  hyphse"  (6). 
Note  the  asexual  reproduction. 

126 


PERISPORACE^E 


I27 


1.  A  filament  grows  upward.     At  its  apex  a  septum 
forms,  then  a  globular  swelling  appears — "sporagium" 
(d).     This  possesses  a  definite  membrane. 

2.  From  the  septum  grows  a  club-shaped  mass  of 
protoplasm — "  columella  "  (c) . 


FIG.  77. — Mucor  mucedo. 


FIG.  78. — Aspergillus 


3.  The  rest  of  the  contained  protoplasm  breaks  up 
into  "  swarm  spores  "  (e) . 

Finally  the  membrane  ruptures  and  spores  escape. 

Perisporaceae. — Aspergillus  (Fig.  78). — Note  the 
branching  filaments — "mycelium"  (a). 


FIG.  79. — Penicillium. 

Note    the    asexual    reproduction. 

i.  A  filament  (b)  grows  upward,  its  termination  be- 
comes clubbed;  on  the  clubbed  extremity  flask-shaped 
cells  appear — "  sterigmata  "  (c) . 


128  CLASSIFICATION    OF    FUNGI 

2.  At  free  end  of  each  sterigma  is  formed  an  oval 
body — a  spore  or  "gonidium"  (d),  which,  when  ripe, 
is  thrown  off  from  the  sterigma.  Two  or  more  gonidia 
may  be  supported  upon  each  sterigma. 

Penicillinm  (Fig.  79). — Note  the  branching  filaments 
— " mycelium"  (a)  (frequently  containing  globules). 

Note  the  asexual  reproduction. 

1.  A  filament  grows  upward — "  goniodophore "   (6) 
— and  its  apex  divides  up  into    several    branches — 
"basidia"  (c). 

2.  At  the  apex  of  each  basidium  a  flask-shaped  cell, 
"sterigma"  (d),  appears. 

3.  At  the  apex  of  each  sterigma  appears  a  row  of 
oval  cells — "spores"  or  "conidia"  (e).     These,  when 
ripe,  are  cast  off  from  the  sterigmata. 

Ascomycetae. — Oldium    (Fig.    80). — (This   family  is 


FIG.  80.— Oidium. 

perhaps  as  nearly  related  to  the  blastomycetes  as  it  is 
to  the  hyphomycetes.) 

Note  the  branching  filaments — "  pseudomycelium  " 
(a).  Here  and  there  filaments  are  broken  up  at  their 
ends  into  oval  or  rod-shaped  segments,  "oidia,"  and 
behave  as  spores. 

Note  the  asexual  reproduction.  From  the  pseudo- 
mycelium  arise  true  hyphae  (6),  each  of  which  in  turn 
ends  in  a  chain  of  spores  (c). 


SACCHAROMYCES  1 29 

MORPHOLOGY  OF  THE  BLASTOMYCETES. 

The  blastomycetes  are  composed  of  spherical  or  ova 
cells  (8  to  9.5  fj.  in  diameter),  which,  when  rapidly 
multiplying  by  budding,  may  form  a  spurious  mycelium. 
A  thin  cell-wall  encloses  the  granular  protoplasm,  in 
which  vacuoles  and  sometimes  a  nucleus  may  be 
noted.  This  latter  is  best  seen  when  stained  with 
haematoxylin  (see  page,  105). 

During  their  growth  and  multiplication  the  blasto- 
mycetes split  up  solutions  containing  sugar  into  alcohol 
and  CO2. 

Saccharomyces  (Fig.  81). — Note  the  round  or  oval 
cells  of  granular  protoplasm  (a)  containing  solid  par- 
ticles and  vacuoles  (<;),  and  surrounded  by  a  definite 
envelope. 

Reproduction. — Budding ;  ascospores — asexual. 

Note  the  asexual  reproduction. 

i.  "Gemmation" — that  is,  the  budding  out  of 
daughter  cells  (b)  from  various  parts  of  the  gradually 
enlarging  mother  cell.  These  are  eventually  cast  off 


FIG.  81. — Saccharomyces  with  ascospores.  FIG.  82. — Torula. 

and  in  turn  become  mother  cells  and  form  fresh  groups 
of  buds. 

2.  Spore  formation — "ascospores"  (e).  These  are 
formed  at  definite  temperatures  and  within  well-de- 
fined periods;  e.  g.,  Saccharomyces  cerevisiae,  thirty 
hours  at  25°  to  37°  C.,  or  ten  days  at  12°  C. 


130  CLASSIFICATION    OF    FUNGI 

Torulse  (Fig.  82). — Torulae  whilst  resembling  yeasts 
in  almost  every  other  respect,  never  form  endo-spores. 
Note  the  elongated,  sausage-shaped  cells  (a)  the  larger 
oval  cells  (6)  and  the  globular  cells  (c)  the  former  two 
often  interlacing  and  growing  as  a  film. 

Note  the  absence  of  ascospore  formation. 


IX.  SCHIZOMYCETES. 

Classification  and  Morphology. — Bacteria  are  often 
classified,  in  general  terms,  according  to  their  life 
functions,  into — 

Saprogenic,  or  putrefactive  bacteria; 
Zymogenic,  or  fermentative  bacteria; 
Pathogenic,  or  disease-producing  bacteria; 

or  according  to  their  food  requirements  into— 

Prototrophic,  requiring  no  organic  food  (e.  g., 

nitrifying  bacteria) ; 
Metatrophic,    requiring   organic   food    (e.   g.t 

saprophytes  and  facultative  parasites) ; 
Paratrophic,   requiring   living  food   (obligate 

parasites) ; 

or  according  to  their  metabolic  products  into— 

Chromogenic,  or  pigment-producing  bacteria; 
Photogenic,  or  light-producing  bacteria ; 
A  erogenic,  or  gas-producing  bacteria; 

and  so  on. 

Such  broad  groupings  as  these  have,  however,  but 
little  practical  value  when  applied  to  the  systematic 
study  of  the  fission  fungi. 

On  the  other  hand,  no  really  scientific  classification 
of  the  schizomycetes  has  yet  been  drawn  up,  and  the 
varying  morphological  appearances  of  the  members 
of  the  family  are  still  utilised  as  a  basis  for  classification, 
as  under — 

1.  Cocci.  (Fig.  83). — Rounded  or  oval  cells,  sub- 
divided according  to  the  arrangement  of  the  individuals 
after  fission,  into — 


I32 


SCHIZOMYCETES 


Diplococci  and  Streptococci,  where  division  takes 
place  in  one  plane  only,  and  the  individuals  remain 
attached  (a)  in  pairs  or  (b)  in  chains. 

Tetrads,  Merismopedia,  or  Pediococci,  where  divi- 
sion takes  place  alternately  in  two  planes  at  right 
angles  to  each  other,  and  the  individuals  remain  at- 
tached in  flat  tablets  of  four,  or  its  multiples. 

$arcin&,  where  division  takes  place  in  three  planes 


123455 

FIG.  83. — Types  of  bacteria — cocci:  i,  Diagram  of  sphere  indicating 
planes  of  fission;  2,  diplococci;  3,  streptococci;  4,  tetrads;  5,  sarcinae;  6, 
staphylococci. 

successively,  and  the  individuals  remain  attached  in 
cubical  packets  of  eight  and  its  multiples. 

Micrococcio?  Staphylococci,  where  division  takes  place 
in  three  planes,  but  with  no  definite  sequence;  conse- 


FIG.   84. — Types  of  bacteria — bacilli,   etc.:  i,   Bacilli 


2,   diplobacilli;  3 


streptobacilli;  4,  spirilla;  5,  vibrios;  6,  spirochaetae. 

quently  the  individuals  remain  attached  in  pairs,  short 
chains,  plates  of  four,  cubical  packets  of  eight,  and 
irregular  masses  containing  numerous  cocci. 

2.  Bacilli  (Fig.  84,  i  to  3)  .—Rod-shaped  cells.     A 
bacillus,  however  short,  can  usually  be  distinguished 


SPIRILLA  133 

from  a  coccus  in  that  two  sides  are  parallel.  Some 
bacilli  after  fission  retain  a  characteristic  arrangement 
and  may  be  spoken  of  as  Diplobacilli  or  Streptobacilli. 

(Leptothrix  is  a  term  that  in  the  past  has  been  loosely 
used  to  signify  a  long  thread,  but  is  now  restricted  to 
such  forms  as  belong-  to  the  leptothricise  (vide  infra) . 

3.  Spirilla  (Fig.  84,  4  to  6). — Curved  and  twisted 
filaments.  Classified,  according  to  shape,  into — 

Spirillum. 
Vibrio  (comma). 
Spirochaeta. 

Many  Spirochaetes  appear  to  belong  to  the  animal 
kingdom  and  are  grouped  under  protozoa ;  other  organ- 
isms to  which  this  name  has  been  given  are  undoubt- 
edly bacteria. 

Higher  forms  of  bacteria  are  also  met  with,  which 
possess  the  following  characteristics:  They  are  at- 
tached, unbranched,  filamentous  forms,  showing — 

(a)  Differentiation  between  base  and  apex; 

(b)  Growth  apparently  apical ; 

(c)  Exaggerated  pleomorphism ; 

(d)  "Pseudo-branching"   from  apposition  of  cells; 
and  are  classified  into — 


1 .  Beggiotoa. 

2.  Thiothrix. 

3.  Crenothrix. 

4.  Cladothrix. 


Free  swimming  forms,  which 
contain  sulphur  granules. 

These   forms  do  not  contain 
sulphur  granules. 


5.  Leptothrix. 

6.  Streptothrix.'    A  group  which  exhibits  true  but 
not  dichotomous  branching,  and  contains  some  patho- 
genic species. 

The  morphology  of  the  same  bacterium  may  vary 
greatly  under  different  conditions. 
3  For  example,  under  one  set  of  conditions  the  .exami- 
nation of  a  pure  cultivation  of  a  bacillus  may  show  a 
short  oval  rod  as  the  predominant  form,  whilst  another 


134  SCHIZOMYCETES 

culture  of  the  same  bacillus,  but  grown  under  different 
conditions,  may  consist  almost  entirely  of  long  fila- 
ments or  threads.  This  variation  in  morphology  is 
known  as  "pleomorphism." 

Some  of  the  factors  influencing  pleomorphism  are : 

1.  The  composition,  reaction,   etc.,   of  the  nutrient 
medium  in  which  the  organism  is  growing. 

2.  The  atmosphere  in  which  it  is  cultivated. 

3.  The  temperature  at  which  it  is  incubated. 

4.  Exposure  to  or  protection  from  light. 

The  various  points  in  the  anatomy  morphology  and 
physiology  of  bacteria  upon  which  stress  is  laid  in  the 
following  pages  should  be  studied  as  closely  as  is  possible 
in  preparations  of  the  micro-organisms  named  in  con- 
nection with  each. 

ANATOMY. 

1.  Capsule    (Fig.    85,    b). — A    gelatinous    envelope 
(probably  akin  to  mucin  in  composition)  surrounding 
each  individual  organism,  and  preventing  absolute  con- 
tact between  any  two.     In  some  species  the  capsule 
(e.  g.,  B.  pneumonias)  is  well  marked,  but  it  cannot  be 
demonstrated  in  all.     In  very  well  marked  cases  of 
gelatinisation  of  the  cell  wall,  the  individual  cells  are 
cemented  together  in  a  coherent  mass,  to  which  the 
term  "zooglcea"  is  applied  (e.  g.,  Streptococcus  mesen- 
teroides) .     In  some  species  colouring  matter  or  ferric 
oxide  is  stored  in  the  capsule. 

2.  Cell   Wall    (Fig.    85,    c). — A  protective   differen- 
tiation  of  the    outer    layer  of  the   cell   protoplasm; 
difficult  to  demonstrate,  but  treatment  with  iodine  or 
salt  solution  sometimes  causes  shrinkage  of  the  cell 
contents — "  plasmolysis  " — and  so  renders  the  cell  wall 
apparent    (e.    g.,    B.   megatherium)    in    the    manner 
shown  in  figure  85.     Stained  bacilli,  when  examined 
with  the  polarising  microscope,  often  show  a  doubly 


STRUCTURE    OF    BACTERIA 


135 


refractile  cell  wall  (e.  g.,  B.  tuberculosis  and  B. 
anthracis) . 

In  some  of  the  higher  bacteria  the  cell  wall  exhibits 
this  differentiation  to  a  marked  degree  and  forms 
a  hard  sheath  within  which  the  cell  protoplasm  is 
freely  movable;  and  during  the  process  of  reproduction 
the  cell  protoplasm  may  be  extruded,  leaving  the 
empty  tube  unaltered  in  shape. 

3.  Cell  Contents. — Protoplasm  (mycoprotein)  con- 
tains a  high  percentage  of  nitrogen,  but  is  said  to  differ 


FIG.  85. — Diagrammatic  sketch  of 
composite  bacterium  to  illustrate 
details  of  anatomical  structure. 


FIG.  86. — Plasmolysis. 


from  proteid  in  that  it  is  not  precipitated  by  C2H60. 
It  is  usually  homogeneous  in  appearance — sometimes 
granular — and  may  contain  oil  globules  or  sap  vacuoles 
(Fig.  85,  d),  chromatin  granules,  and  even  sulphur 
granules.  Sap  vacuoles  must  be  distinguished  from 
spores,  on  the  one  hand,  and  the  vacuolated  appear- 
ance due  to  plasmolysis,  on  the  other. 

The  cell  contents  may  sometimes  be  differentiated 
into  a  parietal  layer,  and  a  central  body  (e.  g.,  beg- 
giotoa)  when  stained  by  haematoxylin. 

4.  Nucleus. — This  structure  has  not  been  conclu- 


136  SCHIZOMYCETES 

sively  proved  to  exist,  but  in  some  bacteria  chromatin 
particles  have  been  observed  near  the  centre  of  the 
bacterial  cell  and  denser  masses  of  protoplasm  situated 
at  the  poles  which  exhibit  a  more  marked  affinity  than 
the  rest  of  the  cell  protoplasm  for  aniline  dyes.  These 
latter  are  termed  polar  granules  or  Polkoerner  (Fig.  85, 
e).  Occasionally  these  aggregations  of  protoplasm 
alter  the  colour  of  the  dye  they  take  up.  They  are 
then  known  as  metachromatic  bodies  or  Ernstschen 
Koerner  (e.  g.,  B.  diphtherias). 

5.  Flagella  (Organs  of  Locomotion,  Fig.  85,   a). — 
These  are  gelatinous  elongations  of  the  cell  protoplasm 

(or  more  probably  of  the  cap- 
sule), occurring  either  at  one 
pole,  at  both  poles,  or  scat- 
tered around  the  entire  periph- 
ery. Flagella  are  not  pseu- 
dopodia.  The  possession  of 
flagella  was  at  one  time  sug- 
,  .,.  V  gested  as  a  basis  for  a  system  of 

FIG.  87. — Types  of  ciliation.  .  .  J 

classification,  when  the  follow- 
ing types  of  ciliation  were  differentiated  (Fig.  87) : 

1.  Polar:  (a)  Monotrichous  (a  single  flagellum  situ- 
ated at  one  pole;  e.  g.,  B.  pyocyaneus). 

(b)  Amphitrichous  (a  single  flagellum  at  each  pole; 
e.  g.,  Spirillum  volutans). 

(c)  Lophotrichous  (a  tuft  or  bunch  of  flagella  situated 
at  each  pole;  e.  g.,  B.  cyanogenus). 

2.  Diffuse:  Peritrichous  (flagella  scattered  around  the 
entire  periphery:  e.  g.,  B.  typhosus). 

PHYSIOLOGY. 

Reproduction. — Active  Stage. — Vegetative,  i.   e.,   by 
the  division  of  cells,  or  "  fission." 

1.  The  cell  becomes  elongated  and  the  protoplasm 
aggregated  at  opposite  poles. 

2.  A   circular   constriction   of   the   organism   takes 


REPRODUCTION  137 

place  midway  between  these  aggregations,  and  a  sep- 
tum is  formed  in  the  interior  of  the  cell  at  right  angles 
to  its  length. 

3 .  The  division  deepens,  the  septum  divides  into  two 
lamellas,     and    finally    two    cells    are    formed. 


cz=:ic 


oOQ 


GOOD 

FIG.  88.  —  Fission  of  cocci.  FIG.  89.  —  Fission  of  bacteria. 

4.  The  daughter  cells  may  remain  united  by  the 
gelatinous  envelope  for  a  variable  time.  Eventually 
they  separate  and  themselves  subdivide. 

Cultures  on  artificial  media, 
after  gro  wing  in  the  same  me- 
dium for  some  time  —  i.  e.,  when 
the  pabulum  is  exhausted  —  show 
"  in  volution  forms"  (Fig.  90), 
well  exemplified  in  cultures  of  B. 
pestis  on  agar  two  days  old,  B. 
diphtherias  on  potato  four  to 
six  days  old. 

They  are  of  two  classes,  viz.  : 

(a)  Involution  forms  charac- 
terised by  alterations  of  shape 

(Fig.     90).        (Not    necessarily   FIG      _^^  forms. 
dead.) 

(b)  Involution  forms  characterised  by  loss  of  staining 
power.     (Always  dead.) 

Resting  Stage.  —  Spore  Formation.  —  Conditions  in- 
fluencing spore  formation:  In  an  old  culture  nothing 
may  be  left  but  spores.  It  used  to  be  supposed  that 
spores  were  always  formed,  so  that  the  species  might 
not  become  extinct,  when 

(a)  The  supply  of  nutrient  was  exhausted. 


138  SCHIZOMYCETES 

(6)  The  medium  became  toxic  from  the  accumula- 
tion of  metabolic  products. 

(c)  The  environment  became  unfavourable;  e.  g., 
change  of  temperature. 

This  is  not  altogether  correct;  e.  g.,  the  temperature 
at  which  spores  are  best  formed  is  constant  for  each 
bacterium,  but  varies  with  different  species;  again, 
aerobes  require  oxygen  for  sporulation,  but  anaerobes 
will  not  spore  in  its  presence. 

(A)  Arthrogenous :  Noted  only  in  the  micrococci. 
One  complete  element  resulting  from  ordinary  fission 
becomes  differentiated  for  the  purpose,  enlarges,  and 
develops  a  dense  cell  wall.     One  or  more  of  the  cells  in  a 
series  may  undergo  this  alteration. 

This  process  is  probably  not  real  spore  formation, 
but  merely  relative  increase  of  resistance.  These  so- 
called  arthrospores  have  never  been  observed  to  "ger- 
minate," nor  is  their  resistance  very  marked,  as  they 
fail  to  initiate  new  cultures,  after  having  been  exposed 
to  a  temperature  of  80°  C.  for  ten  minutes. 

(B)  Endogenous:  The  cell  protoplasm  becomes  dif- 
ferentiated and  condensed  into  a  spherical  or  oval 
mass  (very  rarely  cylindrical) .     After  further  contrac- 
tion the  outer  layers  of  the  mass  become  still  more 
highly  differentiated  and  form  a  distinct  spore  mem- 
brane, and  the  spore  itself  is  now  highly  refractile. 
It  has  been  suggested,  and  apparently  on  good  grounds, 
that  the  spore  membrane  consists  of  two  layers,  the 
exosporium  and  the  endosporium.     Each  cell  forms 
one  spore  only,  usually  in  the  middle,  occasionally  at 
one  end  (some  exceptions,  however,  are  recorded;  e.  g., 
B.  inflatus).     The  shape  of  the  parent  cell  may  be  un- 
altered, as  in  the  anthrax  bacillus,  or  altered,  as  in  the 
tetanus  bacillus,  and  these  points  serve  as  the  basis 
for  a  classification  of  spore-bearing  bacilli,  as  follows: 

(A)  Cell  body  of  the  parent  bacillus  unaltered  in 
shape  (Fig.  91,  a). 


a        b          c          d        e 

PWT 


SPORE    FORMATION 

(B)   Cell  of  the  parent   bacillus  altered  in  shape. 

1.  Clostridium  (Fig.  gi,b):  Rod  swollen  at  the  centre 
and  attenuated  at  the  poles;  spindle  shape;  e.  g.,  B. 
butyricus. 

2.  Cuneate  (Fig.  91,  c) :  Rods  swollen  slightly  at  one 
pole  and  more  or  less  pointed  at  the  other;  wedge- 
shaped. 

3.  Clavate  (Fig.  91,  d) :  Rods 
swollen  at  one  pole  and  cylin- 
drical (unaltered)  at  the  other; 
keyhole-shaped;    e.   g.,    B. 
chauvei. 

4.  Capitate  (Fig.  91,  e) :  Rods  FlG*  91--? 
with  a    spherical  enlargement 

at  one  pole;  drumstick-shaped;  e.  g.,  B.  tetani. 

The  endospores  remain  within  the  parent  cell  for  a 
variable  time  (in  one  case  it  is  stated  that  germination 
of  the  spore  occurs  within  the  interior  of  the  parent  cell 
— "  endo-germination  ") ,  but  are  eventually  set  free, 
as  a  result  of  the  swelling  up  and  solution  of  the  cell 
membrane  of  the  parent  bacillus  in  the  surrounding 
liquid,  or  of  the  rupture  of  that  membrane.  They  then 
present  the  following  characteristics: 

1.  Well-formed,  dense  cell  membranes,  which  renders 
them  extremely  difficult  to  stain,  but  when  once  stained 
equally  difficult  to  decolourise. 

2.  High  refractility,  which  distinguished  them  from 
vacuoles. 

3.  Higher  resistance  than  the  parent  organism  to 
such  lethal   agents   as   heat,    desiccation,    starvation, 
time,  etc.,  this  resistance  being  due  to 

(a)  Low  water  contents  of  plasma  of  the  spore. 

(b)  Low  heat-conducting  power       1  of  the  spore 

(c)  Low  permeability  J  membrane. 
This  resistance  varies  somewhat  with  the  particular 

species — e.  g.,  some  spores  may  resist  boiling  for  a  few 


140  SCfflZOMYCETES 

minutes — but  practically  all  are  killed  if  the  boiling  is 
continued  for  ten  minutes. 

Germination. — When  transplanted  to  suitable  media 
and  placed  under  favourable  conditions,  the  spores 
germinate,  usually  within  twenty-four  to  thirty-six 
hours,  and  successively  undergo  the  following  changes 
which  may  be  followed  in  hanging-drop  cultures  on  a 
warm  stage: 

1.  Swell  up  slowly  and  enlarge,  through  the  absorp- 
tion of  water. 

2.  Lose  their  refrangibility. 

3.  At  this  stage  one  of  three  processes  (but  the  par- 
ticular process  is  always  constant  for  the  same  species) 
may  be  observed: 

(a)  The  spore  grows  out  into  the  new  bacillus 
without  discarding  the  spore  membrane  (which  in 
this  case  now  becomes  the  cell  membrane) ;  e.  g.,  B. 
leptosporus. 

(6)  It  loses  its  spore  membrane  by  solution;  e.  g.,  B. 
anthracis. 

(c)  It  loses  its  spore  membrane  by  rupture. 

In  this  process  the  rupture  may  be  either  polar  -(at 
one  pole  only  e.  g.,  B.  butyricus),  or  bipolar  (e.  g., 
B.  sessile),  or  equatorial;  (e.  g.,  B.  subtilis). 

In  those  cases  where  the  spore  membrane  is  discarded 
the  cell  membrane  of  the  new  bacillus  may  either  be 
formed  from — 

(a)  The  inner  layer  of  the  spore  membrane,  which 
has  undergone  a  preliminary  splitting  into  parietal 
and  visceral  layers;  e.  g.,  B.  butyricus. 

(6)  The  outer  layers  of  the  cell  protoplasm,  which 
become  differentiated  for  that  purpose;  e.  g.,  B.  mega- 
therium. 

The  new  bacillus  now  increases  in  size,  elongates, 
and  takes  on  a  vegetative  growth — i.  e.,  undergoes 
fission — the  bacilli  resulting  from  which  may  in  their 
turn  give  rise  to  spores. 


GERMINATION 


141 


O  0 


FIG.  92.  Simple. 


00 


FIG.  93.  Solution. 


oO 


~*_  ~ 

O 


FIG.  94.  Polar. 


0 


c 


FIG.  95.  Bipolar. 


0 


FIG.  96.  Equatorial. 


142  SCHIZOMYCETES 

Food  Stuffs. — i.  Organic  Foods. — 

(a)  The  pure  parasites  (e.  g.,  B.  leprae)  will  not  live 
outside  the  living  body. 

(6)  Both  saprophytic  and  facultative  parasitic  bac- 
teria agree  in  requiring  non-concentrated  food. 

(c)  The  facultative  parasites  need  highly  organised 
foods;  e.  g.,  proteids  or  other  sources  of  nitrogen  and 
carbon,  and  salts. 

(d)  The  saprophytic  bacteria  are  more  easily  culti- 
vated;.?.  g., 

1.  Some  bacteria  will  grow  in  almost  pure  distilled 
water. 

2.  Some  bacteria  will  grow  in  pure  solutions  of  the 
carbohydrates. 

3.  Water    is   absolutely  essential  to  the   growth  of 
bacteria. 

Food  of  a  definite  reaction  is  needed  for  the  growth 
of  bacteria.  As  a  general  rule  growth  is  most  active 
in  media  which  react  slightly  acid  to  phenolphthalein 
• — that  is,  neutral  or  faintly  alkaline  to  litmus.  Mould 
growth,  on  the  other  hand,  is  most  vigourous  in  media 
that  are  strongly  acid  to  phenolphthalein. 

Environment. — The  influence  of  physical  agents 
upon  bacterial  life  and  growth  is  strongly  marked. 

1.  Atmosphere. — The  presence  of  oxygen  is  necessary 
for  the  growth  of  some  bacteria,  and  death  follows 
when  the  supply  is  cut  off.     Such  organisms  are  termed 
obligate  aerobes. 

Some  bacteria  appear  to  thrive  equally  well  whether 
supplied  with  or  deprived  of  oxygen.  These  are  termed 
facultative  anaerobes. 

A  third  class  will  only  live  and  multiply  when  the 
access  of  free  oxygen  is  completely  excluded.  These 
are  termed  obligate  anaerobes. 

2.  Temperature. — Practically    no    bacterial    growth 
occurs  below  5°  C.,  and  very  little  above  40°  C.     30°  C. 


THE    METABOLISM    OF    BACTERIA  143 

to  3  7°  C.  is  the  most  favorable  for  the  large  majority 
of  micro-organisms. 

The  maximum  and  minimum  temperatures  at  which 
growth  takes  place,  as  well  as  the  optimum,  are  fairly 
constant  for  each  bacterium. 

Bacteria  have  been  classified,  according  to  their 
optimum  temperature,  into— 

MIN.  OPT.          MAX. 

1.  Psychrophilic  bacteria  (chiefly 

water  organisms o°  C.          15°  C.     •     30°  C. 

2.  Mesophilic  bacteria  (includes  patho- 

genic bacteria)     15°  C.          37°  C.          45°  C. 

3.  Thermophilic  bacteria 45°  C.          55°  C.          70°  C. 

The  thermal  death-point  of  an  organism  is  another 
biological  constant;  and  is  that  temperature  which 
causes  the  death  of  the  vegetative  forms  when  the 
exposure  is  continued  for  a  period  of  ten  minutes  (see 
pages  298-301). 

3.  Light. — Many  organisms  are  indifferent  to   the 
presence  of  light.     On  the  other  hand,  light  frequently 
impedes  growth,  and  alters  to  a  greater  or  lesser  extent 
the   biochemical  characters  of  the  organisms — e.   g.y 
chromogenicity  or  power  of  liquefaction.     Pathogenic 
bacteria  undergo  a  progressive  loss  of  virulence  when 
cultivated  in  the  presence  of  light. 

4.  Movements. — Movements,  if  slight  and  simply  of 
a  flowing  character,  do  not  appear  to  injuriously  affect 
the  growth  of  bacteria;  but  violent  agitation,  such  as 
shaking,  absolutely  kills  them. 

A  condition  of  perfect  rest  would  seem  to  be  that 
most  conducive  to  bacterial  growth. 

The  Metabolic  Products  of  Bacteria. — Pigment  Pro- 
duction.— Many  micro-organisms  produce  one  or  more 
vivid  pigments — yellow,  orange,  red,  violet,  fluorescent, 
etc. — during  the  course  of  their  life  and  growth.  The 
colouring  matter  usually  exists  as  an  intercellular 
excrementitious  substance.  Occasionally,  however,  it 


144  SCHIZOMYCETES 

appears  to  be  stored  actually  within  the  bodies  of  the 
bacteria.  The  chromogenic  bacteria  are  therefore 
classified,  in  accordance  with  the  final  destination  of 
the  colouring  matter  they  elaborate,  into — 

Chromoparous  Bacteria:  in  which  the  pigment  is 
diffused  out  upon  and  into  the  surrounding  medium. 

Chromophorous  Bacteria:  in  which  the  pigment  is 
stored  in  the  cell  protoplasm  of  the  organism. 

Parachromophorous  Bacteria:  in  which  the  pigment 
is  stored  in  the  cell  wall  of  the  organism. 

Different  species  of  chromogenic  bacteria  differ  in 
their  requirements  as  to  environment,  for  the  produc- 
tion of  their  characteristic  pigments;  e.  g.,  some  need 
oxygen,  light,  or  high  temperature;  others  again  favor 
the  converse  of  these  conditions. 

Light  Production. — Some  bacteria,  and  usually  those 
originally  derived  from  water,  whether  fresh  or  salt, 
exhibit  marked  phosphorescence  when  cultivated  under 
suitable  conditions.  These  are  classed  as  ' '  photogenic. ' ' 

Enzyme  Production. — Many  bacteria  produce  soluble 
ferments  or  enzymes  during  the  course  of  their  growth, 
as  evidenced  by  the  liquefaction  of  gelatine,  the  clot- 
ting of  milk,  etc.  These  ferments  may  belong  to  either 
of  the  following  well-recognised  classes:  proteolytic, 
diastatic,  invertin,  rennet. 

Toxin  Production. — A  large  number,  especially  of 
the  pathogenic  bacteria,  elaborate  or  secrete  poisonous 
substances  concerning  which  but  little  exact  knowledge 
is  available,  although  many  would  appear  to  be  en- 
zymic  in  their  action. 

These  toxins  are  usually  differentiated  into — 

Extracellular  (or  Soluble)  Toxins:  those  which  are 
diffused  into,  and  held  in  solution  by,  the  surrounding 
medium. 

Intracellular  (or  Inseparate)  Toxins :  those  which  are 
so  closely  bound  up  with  the  cell  protoplasm  of  the 
bacteria  elaborating  them  that  up  to  the  present  time 


THE    METABOLISM    OF    BACTERIA  145 

no  means  has  been  devised  for  their  separation  or  ex- 
traction. 

End-products  of  Metabolism. — Under  this  heading 
are  included — 

Organic  Acids  (e.  g.,  lactic,  butyric,  etc.). 

Alkalies  (e.  g.\  ammonia). 

Aromatic  Compounds  (e.  g.,  indol,  phenol). 

Reducing  Substances  (e.  g.,  those  reducing  nitrates 
to  nitrites). 

Gases  (e.  g.,  sulphuretted  hydrogen,  carbon  dioxide, 
etc.) . 

And  while  the  discussion  of  their  formation,  etc.,  is 
beyond  the  scope  of  a  laboratory  handbook,  the 
methods  in  use  for  their  detection  and  separation 
come  into  the  ordinary  routine  work  and  will  therefore 
be  described  (vide  page  276  et  seq.}. 


xo 


X.  NUTRIENT  MEDIA. 

IN  order  that  the  life  and  growth  of  bacteria  may 
be  accurately  observed  in  the  laboratory,  it  is  neces- 
sary— 

1.  To  isolate  individual  members  of  the  different 
varieties  of  micro-organisms. 

2.  To  cultivate  organisms,  thus  isolated,  apart  from 
other  associated  or  contaminating  bacteria — i.  e.,  in 
pure  culture. 

For  the  successful  achievement  of  these  objects  it  is 
necessary  to  provide  nutriment  in  a  form  suited  to  the 
needs  of  the  particular  bacterium  or  bacteria  under 
observation,  and  in  a  general  way  it  may  be  said  that 
the  nutrient  materials  should  approximate  as  closely 
as  possible,  in  composition  and  character,  to  the  natural 
pabulum  of  the  organism. 

The  general  requirements  of  bacteria  as  to  their 
food-supply  have  already  been  indicated  (page  142) 
and  many  combinations  of  proteid  and  of  carbohy- 
drate have  been  devised,  from  time  to  time,  on  those 
lines.  These,  together  with  various  vegetable  tissues, 
physiological  or  pathological  fluid  secretions,  etc.,  are 
collectively  spoken  of  as  nutrient  media  or  culture  media. 

The  greater  number  of  these  media  are  primarily 
fluid,  but,  on  account  of  the  rapidity  with  which  bac- 
terial growth  diffuses  itself  through  a  liquid,  it  is  im- 
possible to  study  therein  the  characteristics  of  indi- 
vidual organisms.  Many  such  media  are,  therefore, 
subsequently  rendered  solid  by  the  addition  of  sub- 
stances like  gelatine  or  agar,  in  varying  proportions, 
the  proportions  of  such  added  material  being  generally 
mentioned  when  referring  to  the  media;  e.  g.,  10  per 
cent,  gelatine,  2  per  cent.  agar.  Gelatine  is  employed 

146 


NUTRIENT   MEDIA  147 

for  the  solidification  of  those  media  it  is  intended  to 
use  in  the  cultivation  of  bacteria  at  the  room  tem- 
perature or  in  the  "cold"  incubator.  In  the  percent- 
ages usually  employed,  gelatine  media  become  fluid  at 
25°  C.;  higher  percentages  remain  solid  at  somewhat 
higher  temperatures,  but  the  difficulty  of  filtering 
strong  solutions  of  gelatine  militates  against  their  gen- 
eral use. 

Media,  on  the  other  hand  which  have  been  solidified 
by  the  addition  of  agar,  only  become  liquid  when  ex- 
posed to  90°  C.  for  about  ten  minutes,  and  again  solidify 
when  the  temperature  falls  to  40°  C. 

When  it  becomes  necessary  to  render  these  media 
fluid,  heat  is  applied,  upon  the  withdrawal  of  which  they 
again  assume  their  solid  condition.  Such  media  should 
be  referred  to  as  liquefiable  media;  in  point  of  fact, 
however,  they  are  usually  grouped  together  with  the 
solid  media. 

NOTE. — It  must  here  be  stated  that  the  designation  10  per 
cent,  gelatine  or  2  per  cent,  agar  refers  only  to  the  quantity  of 
those  substances  actually  added  in  the  process  of  manufacture, 
and  not  to  the  percentage  of  gelatine  or  agar,  as  the  case  may  be, 
present  in  the  finished  medium;  the  explanation  being  that  the 
commercial  products  employed  contain  a  large  proportion  of 
insoluble  material  which  is  separated  off  by  filtration  during  the 
preparation  of  the  liquefiable  media. 

Other  media,  again — e.  g.,  potato,  coagulated  blood- 
serum,  etc. — cannot  be  again  liquefied  by  physical 
means,  and  these  are  spoken  of  as  solid  media. 

The  following  pages  detail  the  method  of  preparing 
the  various  nutrient  media,  in  ordinary  use  (see  also 
Chapter  XI),  those  which  are  only  occasionally  required 
for  more  highly  specialised  work  are  grouped  together 
in  Chapter  XII.  It  must  be  premised  that  scrupulous 
cleanliness  is  to  be  observed  with  regard  to  all  ap- 
paratus, vessels,  funnels,  etc.,  employed  in  the  prepara- 
tion of  media;  although  in  the  preliminary  stages  of 


148  NUTRIENT   MEDIA 

the  preparation  of  most  media  absolute  sterility  of  the 
apparatus  used  is  not  essential. 


MEAT  EXTRACT. 

A  watery  solution  of  the  extractives,  etc.,  of  lean 
meat  (usually  beef)  forms  the  basis  of  several  nutrient 
media.  This  solution  is  termed  "  meat  extract "  and  it 
has  been  determined  empirically  that  its  preparation 
shall  be  carried  out  by  extracting  half  a  kilo  of  moist 
meat  with  one  litre  of  water.  For  many  purposes, 
however,  it  is  more  convenient  to  have  a  more  concen- 
trated extract;  one  kilo  of  meat  should  therefore  be 
extracted  with  one  litre  of  water,  to  form  "  Double 
Strength"  meat  extract. 

It  was  customary  at  one  time,  and  is  even  now  in 
some  laboratories  to  use  either  "  shin  of  beef"  or  "beef- 
steak"— both  contain  muscle  sugar  which  often  needs 
to  be  removed  before  the  nutrient  medium  can  be 
completed.  Heart  muscle  (bullock's  heart  or  sheep's 
heart)  is  much  to  be  preferred  and  from  the  point  of 
economy,  ease  and  cleanliness  of  manipulation,  and 
extractive  value,  the  imported  frozen  bullock's  hearts 
provide  the  best  extract. 

Meat  extract  (Fleischwasser)  is  prepared  as  follows: 

1.  Measure  1000  c.c.  of  distilled  water  into  a  large 
flask  (or  glass  beaker,  or  enamelled  iron  pot)  and  add 
1000  grammes    (roughly,    2j    pounds)    of    fresh  lean 
meat — e.    g.t    bullock's    heart — finely    minced    in    a 
mincing  machine. 

2.  Heat  the  mixture  gently  in  a  water-bath,  taking 
care  that  the  temperature  of  the  contents  of  the  flask 
does  not  exceed  40°  C.  for  the  first  twenty  minutes. 
(This  dissolves  out  the  soluble  proteids,  extractives, 
salts,  etc.) 

3 .  Now  raise  the  temperature  of  the  mixture  to  the 
boiling-point,   and  maintain  at  this  temperature  for 


REACTION    OF    MEAT    EXTRACT  149 

ten  minutes.     (This  precipitates  some  of  the  albumins, 
the  haemoglobin,  etc.,  from  the  solution.) 

4.  Strain  the  mixture  through  sterile  butter  muslin 
or  a  perforated  porcelain  funnel,  then  filter  the  liquid 
through  Swedish  filter  paper  into  a  sterile  "normal" 
litre  flask,  and  when  cold  make  up  to  1000  c.c.  by 
the  addition  of  distilled  water — to  replace  the  loss  from 
evaporation. 

5 .  If  not  needed  at  once,  sterilise  the  meat  extract  in 
bulk  in  the  steam  steriliser  for  twenty  minutes  on  each 
of  three  consecutive  days. 

Calf,  sheep,  or  chicken  flesh  is  occasionally  substi- 
tuted for  the  beef;  or  the  meat  extract  may  be  pre- 
pared from  animal  viscera,  such  as  brain,  spleen,  liver, 
or  kidneys. 

NOTE. — As  an  alternative  method,  5  c.c.  of  Brand's  meat  juice 
or  3  grammes  of  Wyeth's  beef  juice,  or  10  grammes  Liebig's 
extract  of  meat  (Lemco)  may  be  dissolved  in  1000  c.c.  distilled 
water,  and  heated  and  filtered  as  above  to  form  ordinary  or 
single  strength  meat  extract. 

Media,  prepared  from  such  meat  extracts  are,  however, 
eminently  unsatisfactory  when  used  for  the  cultivation  of  the 
more  highly  parasitic  bacteria;  although  when  working  in  tropical 
and  subtropical  regions  their  use  is  well-nigh  compulsory. 

Reaction  of  Meat  Extract. — Meat  extract  thus  pre- 
pared is  acid  in  its  reaction,  owing  to  the  presence  of 
acid  phosphates  of  potassium  and  sodium,  weak  acids 
of  the  glycolic  series,  and  organic  compounds  in  which 
the  acid  character  predominates.  Owing  to  the  nature 
of  the  substances  from  which  it  derives  its  reaction, 
the  total  acidity  of  meat  extract  can  only  be  estimated 
accurately  when  the  solution  is  at  the  boiling-point. 

Moreover,  it  has  been  observed  that  prolonged  boiling 
(such  as  is  involved  in  the  preparation  of  nutrient 
media)  causes  it  to  undergo  hydrolytic  changes  which 
increase  its  acidity,  and  the  meat  extract  only  becomes 
stable  in  this  respect  after  it  has  been  maintained  at 
the  boiling=point  for  forty =five  minutes. 


I5O  NUTRIENT   MEDIA 

Although  meat  extract  always  reacts  acid  to  phenol- 
phthalein,  it  occasionally  reacts  neutral  or  even  alka- 
line to  litmus;  and  again,  meat  extract  that  has  been 
rendered  exactly  neutral  to  litmus  still  reacts  acid  to 
phenolphthalein.  This  peculiar  behaviour  depends 
upon  two  factors : 

1 .  Litmus  is  insensitive  to  many  weak  organic  acids 
the  presence  of  which  is  readily  indicated  by  phenol- 
phthalein. 

2.  Dibasic  sodium  phosphate  which  is  formed  during 
the  process  of  neutralisation  is  a  salt  which  reacts 
alkaline  to   litmus,    but   neutral   to   phenolphthalein. 
In  order,  therefore,  to  obtain  an  accurate  estimation 
of  the  reaction  of  any  given  sample  of  meat  extract, 
it  is  essential  that — 

1.  The  meat  extract  be  previously  exposed  to  a  tem- 
perature of  100°  C.  for  forty-five  minutes. 

2.  The  estimation  be  performed  at  the  boiling-point. 

3 .  Phenolpthalein  be  used  as  the  indicator. 

The  estimation  is  carried  out  by  means  of  titration 
experiments  against  standard  solutions  of  caustic  soda, 
in  the  following  manner : 

Method  of  Estimating  the  Reaction. — 

Apparatus  Required:  Solutions  Required: 

1.  25  c.c.  burette  graduated  i.   xoN      NaOH,      accurately 
in  tenths  of  a  centimetre.  standardised. 

2.  i  c.c.  pipette  graduated  in  2.  B  NaOH,  accurately  stand- 
hundredths,  and  provided  .    *     •,•      -, 

with  rubber  tube,  pinch- 
cock,  and  delivery  nozzle.  n 

3.  2 5  c.c.  measure  (cylinder or  3-  r0NaOH'  accurately  stand- 
pipette,  calibrated  for  98°  ardised. 

C.—not  15°  C.).  4>  per    cent     solution    of 

4.  Several     60     c.c.     conical  phenolphthalein  in  50  per 
beakers     or     Erlenmeyer  cent,  alcohol. 

flasks. 

5.  White  porcelain  evaporat- 
ing basin,  filled  with  boil- 
ing water  and   arranged 


TITRATING    MEAT    EXTRACT 


Apparatus  Required:  —  (Continued.') 

over    a    gas    flame    as    a 
water-bath. 

6.  Bohemian  glass  flask, 
.fitted  as  a  wash-bottle, 
and  filled  with  distilled 
water,  which  is  kept  boil- 
ing on  a  tripod  stand. 


METHOD.  —  Arrange   the   apparatus   as   indicated  in 
figure  97. 

(A)    i.  Fill  the  burette  with  ^0  NaOH. 
2.  Fill  the  pipette  with  -f  NaOH. 


FIG.  97.— 


Arrangement  of  apparatus  for  titrating  media. 


3.  Measure  25  c.c.  of  the  meat  extract  (previously 
heated  in  the  steamer  at  100°  C.  for  forty-five  minutes) 
into  one  of   the  beakers   by  means  of  the  measure; 
rinse  out  the  measure  with  a  very  small  quantity  of  boil- 
ing distilled  water  from  the  wash-bottle,  and  then  add 
this  rinse  water  to  the  meat  extract  already  in  the 
beaker. 

4.  Run  in  about  0.5  c.c.  of  the  phenolphthalein  solu- 
tion and  immerse  the  beaker  in  the  water-bath,  and 
raise  to  the  boil. 

5.  To  the  medium  in  the  beaker  run  in  £  NaOH 
cautiously  from   the   burette  until  the  end-point  is 
reached,  as  indicated  by  the  development  of  a  pinkish 


152  NUTRIENT   MEDIA 

tinige,  shown  in  figure  98   (6).     Note  the  amount  of 
decinormal  soda  solution  used  in  the  process. 

NOTE. — Just  before  the  end-point  is  reached,  a  very  slight 
opalescence  may  be  noted  in  the  fluid,  due  to  the  precipitation 
of  dibasic  phosphates.  After  the  true  end-point  is  reached,  the 
further  addition  of  about  0.5  c.c.  of  the  decinormal  soda  solution 
will  produce  a  deep  magenta  colour  (Fig.  98,  c),  which  is  the  so- 
called  "end-point"  of  the  American  Committee  of  Bacteriologists. 


a  l>  c  . 

FIG.  98, — a,  Sample  of  filtered  meat  extract  or  nutrient  gelatine  to  which 
phenolphthalein  has  been  added.  The  medium  is  acid,  as  evidenced  by  the 
unaltered  colour  of  the  sample.  6,  The  same  neutralised  by  the  addition  of 
—  NaOH.  The  production  of  this  faint  rose-pink  colour  indicates  that  the 
"  end-point,"  or  neutral  point  to  phenolphthalein,  has  been  reached.  If  such 
a  sample  is  cooled  down  to  say  30°  or  20°  C.,  the  colour  will  be  found  to 
become  more  distinct  and  decidedly  deeper  and  brighter,  resembling  that 
shown  in  c.  c,  Also  if,  after  the  end-point  is  reached,  a  further  0.5  c.c.  or 
i.o  c.c.  •«  NaOH  be  added  to  the  sample,  the  marked  alkalinity  is  evidenced 
by  the  deep  colour  here  shown. 

(B)  Perform  a  ''control"  titration  (occasionally  two 
controls  may  be  necessary),  as  follows: 

1.  Measure  25  c.c.  of  the  meat  extract  into  one  of  the 
beakers,   wash  out  the  measure  with  boiling  water, 
and  add  the  phenolphthalein  as  in  the  first  estimation. 

2.  Run  in  -f  NaOH  from  the  pipette,  just  short  of 
the   equivalent   of   the   amount   of  deci-normal  soda 
solution  required  to  neutralise  the  2  5  c.c.  of  medium. 
(For  example,   if  in  the  first  estimation  5  c.c.  of  ~ 
NaOH   were  required  to  render   25   c.c.   of  medium 
neutral    to    phenolphthalein,    only    add   0.48    c.c.   of 
7  NaOH.)     Immerse  the  beaker  in  the  water-bath. 

3.  Complete  the  titration  by  the  aid  of  the  £  NaOH. 


REACTION    OF    MEAT   EXTRACT  153 

4.  Note  the  amount  of  ~  NaOH  solution  required 
to  complete  the  titration,  and  add  it  to  the  equivalent 
of  the  -f  NaOH  solution  previously  run  in.  Take  the 
total  as  the  correct  estimation. 

Method  of  Expressing  the  Reaction. — 

The  reaction  or  litre  of  meat  extract,  medium,  or  any 
solution  estimated  in  the  foregoing  manner,  is  most 


FIG.  99. — Stock  bottle  for  deka-normal  soda  solution. 

conveniently  expressed  by  indicating  the  number  of 
cubic  centimetres  of  normal  alkali  (or  normal  acid) 
that  would  be  required  to  render  one  litre  of  the  solution 
exactly  neutral  to  phenolphthalein. 

The  sign  +  (plus)  is  prefixed  to  this  number  if  the 
original  solution  reacts  acid,  and  the  sign  -  (minus)  if 
it  reacts  alkaline. 


154  NUTRIENT   MEDIA 

For  example,  ''meat  extract  +  10, "  indicates  a 
sample  of  meat  extract  which  reacts  acid  to  phenol- 
phthalein,  and  would  require  the  addition  of  10  c.c.  of 
normal  NaOH  per  litre,  to  neutralise  it. 

NOTE. — Such  a  solution  would  probably  react  alkaline  to 
litmus. 

Conversely,  if  as  the  result  of  our  titration  experi- 
ments we  find  that  25  c.c.  of  meat  extract  require  the 
addition  of  5  c.c.  ~  NaOH  to  neutralise,  then  1000  c.c. 
of  meat  extract  will  require  the  addition  of  200  c.c.  ~ 
NaOH  =20  c.c.  -f  NaOH. 

And  this  last  figure,  20,  preceded  by  the  sign  + 
(i.e.,  -f-  20),  to  signify  that  it  is  acid,  indicates  the  re- 
action of  the  meat  extract. 

NOTE. — The  standard  soda  solutions  should  be  prepared  by 
accurate  measuring  operations,  controlled  by  titrations,  from  a 
stock  solution  of  zoN  NaOH,  which  should  be  very  carefully 
standardised.  If  a  large  supply  is  made  or  the  consumption  is 
small  this  stock  solution  must  be  kept  in  an  aspirator  bottle  to 
which  air  can  only  gain  access  after  it  has  been  dried  and  rendered 
free  from  CO2.  This  may  be  done  by  first  leading  it  over  H2SO4 
and  soda  lime,  or  soda  lime  alone,  by  some  such  arrangement  as 
is  shown  in  figure  QQ,  which  also  shows  a  constant  burette  arrange- 
ment for  the  delivery  of  small  measured  quantities  of  the 
dekanormal  soda  solution. 

STANDARDISATION  OF  MEDIA. 

Differences  in  the  reaction  of  the  medium  in  which 
it  is  grown  will  provoke  not  only  differences  in  the  rate 
of  growth  of  any  given  bacterium,  but  also  well-marked 
differences  in  its  cultural  and  morphological  characters ; 
and  nearly  every  organism  will  be  found  to  affect  a 
definite  "optimum  reaction" — a  point  to  be  carefully 
determined  for  each.  For  most  bacteria,  however,  the 
"optimum"  usually  approximates  fairly  closely  to 
-f- 10 ;  and  as  experiment  has  shown  that  this  reaction  is 
the  most  generally  useful  for  routine  laboratory  work, 
it  is  the  one  which  may  be  adopted  as  the  standard  for 
all  nutrient  media  derived  from  meat  extract. 


STANDARDISING    BOUILLON  155 

Briefly,  the  method  of  standardising  a  litre  of  media 
to  +10  consists  in  subtracting  10  from  the  initial  litre 
of  the  medium  mass;  the  remainder  indicates  the 
number  of  cubic  centimetres  of  normal  soda  solution 
that  must  be  added  to  the  medium,  per  litre,  to  render 
the  reaction  +10. 

Standardising  Nutrient  Bouillon. — For  example,  1000 
c.c.  bouillon  are  prepared;  at  the  first  titration  it  is 
found 

1.  25  c.c.  require  the  addition  of  5.50  c.c.  £  NaOH 
to  neutralise. 

Two  controls  give  the  following  results: 

2.  25  c.c.  require  the  addition  of  5.70  c.c.  ~  NaOH 
to  neutralise. 

3.  25  c.c.  require  the  addition  of  5.60  c.c.  ^  NaOH 
to  neutralise. 

Averaging  these  two  controls,  25  c.c.  require  the 
addition  of  5.65  c.c.  ~  NaOH  to  neutralise,  and  there- 
fore 1000  c.c.  require  the  addition  of  226  c.c.  ~  NaOH, 
or  22.60  c.c.  -f  NaOH,  or  2.26  c.c.  10  or  NaOH. 

Initial  litre  of  the  bouillon  =  +22.6,  and  as  such 
requires  the  addition  of  (22.6  c.c.  — 10  c.c.)  =  12. 6  c.c. 
of  -f-  NaOH  per  litre  to  leave  its  finished  reaction  +  10. 

But  the  three  titrations,  each  on  25  c.c.  of  medium, 
have  reduced  the  original  bulk  of  bouillon  to  (1000 
—  75  c.c.)  =  925  c.c.  The  amount  of  -f  NaOH  required 
to  render  the  reaction  of  this  quantity  of  medium  +10 
may  be  deduced  thus : 

1000  c.c.  :  925  c.c.  ::  12.6  c.c.  :x. 

Then*  =11.65  c.c.  -f-  NaOH. 

Whenever  possible,  however,  the  required  reaction 
is  produced  by  the  addition  of  dekanormal  soda  solu- 
tion, on  account  of  the  minute  increase  it  causes  in  the 
bulk,  and  the  consequent  insignificant  disturbance  of 
the  percentage  composition  of  the  medium.  By  means 
of  a  pipette  graduated  to  o.oi  c.c.  it  is  possible  to  de- 


156  NUTRIENT   MEDIA 

liver  very  small  quantities ;  but  if  the  calculated  amount 
runs  into  thousandth  parts  of  a  cubic  centimetre,  these 
are  replaced  by  corresponding  quantities  of  normal  or 
even  decinormal  soda. 

In  the  above  example  it  is  necessary  to  add  11.65 
c.c.  normal  NaOH  or  its  equivalent,  1.165  c-c-  deka- 
normal  NaOH.  The  first  being  too  bulky  a  quantity, 
and  the  second  inconveniently  small  for  exact  measure- 
ment, the  total  weight  of  soda  is  obtained  by  substi- 
tuting 1. 1 6  c.c.  dekanormal  soda  solution,  and  either 
0.05  c.c.  of  normal  soda  solution  or  0.5  c.c.  of  deci- 
normal soda  solution. 

Standardising  Nutrient  Agar  and  Gelatine. — The 
method  of  standardising  agar  and  gelatine  is  precisely 
similar  to  that  described  under  bouillon. 

THE  FILTRATION  OF  MEDIA. 

Fluid  media  are  usually  filtered  through  stout  Swed- 
ish filter  paper  (occasionally  through  a  porcelain  filter 
candle) ,  and  in  order  to  accelerate  the  rate  of  filtration 
the  filter  paper  should  be  folded  in  that  form  which 
is  known  as  the  "physiological  filter,"  not  in  the  ordi- 
nary " quadrant"  shape,  as  by  this  means  a  large 
surface  is  available  for  filtration  and  a  smaller  area  in 
contact  with  the  glass  funnel  supporting  it. 

To  fold  the  filter  proceed  thus : 

1 .  Take  a  circular  piece  of  filter  paper  and  fold  it  ex- 
actly through  its  centre  to  form  a  semicircle  (Fig.  ioo,a). 

2 .  Fold  the  semicircle  exactly  in  half  to  form  a  quad- 
rant ;  make  the  crease  2,  distinct  by  running  the  thumb- 
nail along  it,  then  open  the  filter  out  to  a  semicircle 
again. 

3.  Fold  each  end  of  the  semicircle  in  to  the  centre 
and  so  form  another  quadrant;  smooth  down  the  two 
new  creases  3  and  3  a,  thus  formed  and  again  open  out 
to  a  semicircle. 


FOLDING    FILTERS  157 

4.  The  semicircle  now  appears  as  in  figure  100,  a,  the 
dark  lines  indicating  the  creases  already  formed. 

5.  Fold  the  point  i  over  to  the  point  3,  and  i  a  to  3  a, 
to  form  the  creases  4  and  4a,  indicated  in  the  diagram 
by  the  light  lines.     Fold  point  i  over  to  3<z,  and  la  to  3, 
to  form  the  creases  5  and  5 a. 

6.  Thus  far  the  creases  have  all  been  made  on  the 
same  side  of  the  paper.     Now  subdivide  each  of  the 


FIG.  100. — Filter  folding:  a,  Filter  folded  in  half,  showing  creases;   6, 
appearance  of  filter  on  completion  of  folding;  c,  filter  opened  out  ready  for 


eight  sectors  by  a  crease  through  its  centre  on  the  op- 
posite side  of  the  paper,  indicated  by  the  faint  broken 
lines  in  the  diagram.  Fold  up  the  filter  gradually  as 
each  crease  is  made,  and  when  finished  the  filter  has 
assumed  the  shape  of  a  wedge,  as  in  figure  100,  b. 


158  NUTRIENT   MEDIA 

When  opened  out  the  filter  assumes  the  shape  repre- 
sented in  figure  100,  c. 

The  folded  filter  is  next  placed  inside  a  glass  funnel 
supported  on  a  retort  stand,  and  moistened  with  hot 
distilled  water  before  the  filtration  of  the  medium  is 
commenced. 

Liquef  iable  solid  media  are  filtered  through  a  specially 
made  filter  paper — "papier  Chardin" — which  is  sold 
in  boxes  of  twenty-five  ready-folded  filters. 

Gelatine,  when  properly  made,  filters  through  this 


FIG.  101. — Hot- water  filter  funnel  and  ring  burner. 

paper  as  quickly  as  bouillon  does  through  the  Swedish 
filter  paper,  and  does  not  require  the  use  of  the  hot- 
water  funnel. 

Agar,  likewise,  if  properly  made,  filters  readily, 
although  not  at  so  rapid  a  rate  as  gelatine.  If  badly 
"egged,"  and  also  during  the  winter  months,  it  is 
necessary  to  surround  the  glass  funnel,  in  which  the 
filtration  of  the  agar  is  carried  on,  by  a  hot- water 
jacket.  This  is  done  by  placing  the  glass  funnel  inside 
a  double- walled  copper  funnel — the  space  between  the 


STORING    MEDIA  159 

walls  being  filled  with  water  at  about  90°  C. — and 
supporting  the  latter  on  a  ring  gas  burner  fixed  to  a 
retort  stand  (Fig.  101).  The  gas  is  lighted  and  the 
water  jacket  maintained  at  a  high  temperature  until 
filtration  is  completed.  If  the  steam  steriliser  of  the 
laboratory  is  sufficiently  large,  it  is  sometimes  more 
convenient  to  place  the  flask  and  filtering  funnel  bodily 
inside,  close  the  steriliser  and  allow  filtration  to  proceed 
in  an  atmosphere  of  live  steam,  than  to  use  the  gas  ring 
and  hot- water  funnel. 

STORING  MEDIA  IN  BULK. 

After  filtration  fill  the  medium  into  sterile  litre  flasks 
with  cotton-wool  plugs  and  sterilise  in  the  steamer  for 
twenty  minutes  on  each  of  three 
consecutive  days.  After  the 
third  sterilisation,  and  when  the 
flasks  and  contents  are  cool,  cut 
off  the  top  of  the  cotton-wool 
plug  square  with  the  mouth  of 
the  flask;  push  the  plug  a  short 
distance  down  into  the  neck  of  a  b 

r.      ..  ..    ^-M    .  . Jt  1,1        FIG.     102. — Rubber    cap 

the  flask  and  fill  in  with  melted  closing  store  bottle.  a>  be. 
paraffin  wax  to  the  level  of  the  fore> and  b>  after  sterilizing, 
mouth.  When  the  wax  has  set  the  flasks  are  stored 
in  a  cool  dark  cupboard  for  future  use. 

This  plan  is  not  absolutely  satisfactory,  although 
very  generally  employed  on  occasion,  and  it  is  prefer- 
able to  fill  the  medium  into  long-necked  flint  glass 
bottles  (the  quart  size,  holding  nearly  1000  c.c.,  such  as 
those  in  which  Pasteurised  milk  is  retailed)  and  to 
close  the  neck  of  the  bottle  by  a  special  rubber  cap.1 
This  cap  is  made  of  soft  rubber,  the  lower  part, 
dome-shaped  with  thin  walls,  being  slipped  over  the 
neck  of  the  bottle  (Fig.  102,0).  The  upper  part  is  solid, 

1  This  rubber  cap  has  been  made  for  me  by  the  Holborn  Surgical  Instru- 
ment Co.,  Thavies  Inn,  London,  W.  C. 


l6o  NUTRIENT   MEDIA 

but  with  a  sharp  clean-cut  (made  with  a  cataract  or 
tenotomy  knife)  running  completely  through  its  axis 
from  the  centre  of  the  disc  to  the  top  of  the  dome. 
During  sterilisation  the  air  in  the  neck  of  the  bottle, 
expanded  by  the  heat,  is  driven  out  through  the  valvu- 
lar aperture  in  the  solid  portion  of  the  stopper.  On 
removing  the  bottle  from  the  steam  chamber,  the 
liquid  contracts  as  it  cools,  and  the  pressure  of  the 
external  air  drives  the  solid  piece  of  rubber  down  into 
the  neck  of  the  bottle,  and  forces  together  the  lips  of  the 
slit  (Fig.  102,6).  Thus  sealed,  the  bottle  will  preserve  its 
contents  sterile  for  an  indefinite  period  without  loss 
from  evaporation. 

TUBING  NUTRIENT  MEDIA. 

After  the  final  filtration,  the  nutrient  medium  is 
usually  "tubed" — i.  e.,  filled  into  sterile  tubes  in  defi- 
nite measured  quantities,  usually  10  c.c.  This  process 
is  sometimes  carried  out  by  means  of  a  large  separator 
funnel  fitted  with  a  "three- way"  tap  which  communi- 
cates with  a  small  graduated  tube  (capacity  20  c.c. 
and  graduated  in  cubic  centimetres)  attached  to  the  side. 
The  shape  of  this  piece  of  apparatus,  known  as  Treskow's 
funnel,  renders  it  particularly  liable  to  damage.  It  is 
better,  therefore,  to  arrange  a  less  expensive  piece  of 
apparatus  which  will  serve  the  purpose  equally  well 
(Fig.  103). 

A  Geissler's  three-way  stop-cock  has  the  tube  on  one 
side  of  the  tap  ground  obliquely  at  its  extremity,  and 
the  tube  on  the  opposite  side  cut  off  within  3  cm.  of  the 
tap.  The  short  tube  is  connected  by  means  of  a  per- 
forated rubber  cork  with  a  10  cm.  length  of  stout  glass 
tubing  (1.5  cm.  bore).  The  third  channel  of  the  three- 
way  tap  is  connected,  by  means  of  rubber  tubing,  with 
the  nozzle  of  an  ordinary  separator  funnel.  Finally, 
the  receiving  cylinder  above  the  three-way  tap  is  gradu- 


STORING  "TUBED"  MEDIA 


161 


ated  in  cubic  centimetres  up  to  20,  by  pouring  into  it 
measured  quantities  of  water  and  marking  the  various 
levels  on  the  outside  with  a  writing  diamond. 

Fluid  media  containing  carbohydrates  are  filled  into 
fermentation  tubes  (vide  Fig.  21);  or  into  ordinary 
media  tubes  which  already  have  smaller  tubes,  inverted, 
inside  them  (Fig.  104),  to  collect  the  products  of  growth 
of  gas-forming  bacteria.  When  first  filled,  the  small 
tubes  float  on  the  surface  of  the  medium  •  after  the  first 


FIG.  103.— Separately  funnel  and  three-way  tap  arranged  for  tubing  media. 
FIG.  104. — Gas  tube  (Durham). 

sterilisation  nearly  all  the  air  is  replaced  by  the  medium, 
and  after  the  final  sterilisation  the  gas  tubes  will  be 
submerged  and  completely  filled  with  the  medium. 
Storing  "Tubed"  Media.— Media  after  being  tubed 
are  best  stored  by  packing,  in  the  vertical  position, 
in  oblong  boxes  having  an  internal  measurement  of 
37  cm.  long  by  12  cm.  wide  by  10  cm.  deep.  Each 
box  (Fig.  105)  has  a  movable  partition  formed  by  the 


1 62  MEDIA    BOXES 

vertical  face  of  a  weighted  triangular  block  of  wood, 
sliding  free  on  the  bottom  (Fig.  105,  A) ;  or  by  a  flat 
piece  of  wood  sliding  in  a  metal  groove  in  the  bottom 
of  the  box,  which  can  be  fixed  at  any  spot  by  tightening 
the  thumbscrew  of  a  brass  guide  rod  which  transfixes 
the  partition  (Fig.  105,  B) .  The  front  of  the  box  is  pro- 


FIG.  105 . — Medium  box,  showing  alternative  partitions  A  and  B. 

vided  with  a  handle  and  a  celluloid  label  for  the  name 
of  the  contained  medium.  These  boxes  are  arranged 
upon  shelves  in  a  dark  cupboard — or  preferably  an  iron 
safe — which  should  be  rendered  as  nearly  air-tight  as 
possible,  and  should  have  the  words  " media  stores" 
painted  on  its  doors. 


XI.  CULTURE  MEDIA. 

ORDINARY  OR  STOCK  MEDIA. 

Nutrient  Bouillon.— 

1 .  Measure  out  double  strength  meat  extract,  5000.0., 
into  a  litre  flask  and  add   300    c.c.   distilled   water. 

2.  Weigh  out  Witte's  peptone,  10  grammes  (=  i  per 
cent.),  salt,  5  grammes  (=0.5  per  cent.),  and  mix  into 
a  smooth  paste  with  200  c.c.  of  distilled  water  pre- 
viously heated  to  60°  C.     (Be  careful  to  leave  no  un- 
broken globular  masses  of  peptone.) 

3.  Add  the  peptone  emulsion  to  the  meat  extract  in 
the  flask  and  heat  in  the  steamer  for  forty-five  minutes 
(to  completely  dissolve  the  peptone,  and  to  render  the 
acidity  of  the  meat  extract  stable) . 

4.  Estimate  the  reaction  of  the  medium;  control  the 
result;  render  the  reaction  of  the  finished  medium 
+  10  (vide  page  155). 

5.  Heat  for  half  an  hour  in  the  steamer  at   100° 
C.   (to  complete  the  precipitation  of  the  phosphates, 
etc.). 

6.  Filter  through  Swedish  filter  paper  into  a  sterile 
flask. 

7.  Fill   into   sterile   tubes    (10   c.c.   in   each  tube). 

8.  Sterilise  in  the  steamer  for  twenty  minutes  on 
each  of  three  consecutive  days — i.  e.t  by  the  discon- 
tinuous method  (vide  page  35). 

NOTE. — As  an  alternative  method  when  neither  fresh  nor  frozen 
meat  is  available  nutrient  bouillon  may  be  prepared  from  a  com- 
mercial meat  extract,  as  follows: 

Lemco  Broth. — 

1.  Measure  out  250  c.c.  distilled  water  into  a  litre  flask. 

2.  Weigh  out  10  grammes  Liebig's  Lemco  Meat  Extract  on  a 

163 


164 


CULTURE    MEDIA 


piece  of  clean  filter  paper  and  add  to  the  water  in  the  flask. 
Shake  the  flask  well  to  make  an  even  emulsion  of  the  meat 
extract. 

3.  Weigh  out  Witte's  peptone  (10  grammes),  salt  (5  grammes). 
Mix  into  smooth  paste  with  100  c.c.  distilled  water  previously 
heated  to  60°  C. 

5.  Add  the  peptone  salt  emulsion  to  the  meat  extract  emulsion 
in  the  flask  and  add  650  c.c.  distilled  water.      Heat  in  the  steamer 
for  forty-five  minutes. 

6.  Standardise    the    medium    and    complete    as    for    nutrient 
bouillon. 

Nutrient  Gelatine.— 

i.  Weigh  a  2 -litre  flask  on  a  trip  balance  (Fig.  106) 
and  note  the  weight,  or  counterpoise  carefully. 


FIG.  106. — Trip  balance. 

An  extremely  useful  counterpoise  is  a  small  sheet - 
brass  cylinder  about  38  mm.  high  and  38  mm.  in  di- 


FIG.  107. — Counterpoise;  weight  when  empty,  35  grammes;  when  full  of  dust 
shot,  200  grammes. 

ameter,  with  a  funnel-shaped  top  and  provided  with  a 
side  tube  by  which  its  contents,  fine  "dust"  shot,  may 
be  emptied  out  (Fig.  107). 


DISSOLVING   GELATINE 


2 .  Measure  out  double  strength  meat  extract,  500  c.c., 
into  the  "tared"  flask. 

3.  Weigh  out  and  mix  10  grammes  of  peptone,   5 
grammes  of  salt,  and  make  into  a  thick  paste  with  150 
c.c.  distilled  water;  then  add  the  emulsion  to  the  meat 
extract  in  the  flask ;  also  add  100  grammes  sheet  gelatine 
cut  into  small  pieces;  place  the  flask  in  the  water- 
bath  and  raise  to  the  boil. 


FIG.  108. — Arrangement  of  steam  can  and  water-bath  for  the  preparation 

of  media. 

4.  Arrange  a  5-litre  tin  can  (with  copper  bottom, 
such  as  is  used  in  the  preparation  of  distilled  water) 
by  the  side  of  the  water  bath,  fill  the  can  with  boiling 
water  and  place  a  lighted  Bunsen  burner  under  it. 
Fit  a  long  safety  tube  to  the  neck  of  the  can  and 
also  a  delivery  tube,  bent  twice  at  right  angles; 
adjust  the  tube  to  reach  to  the  bottom  of  the  interior 
of  the  flask  containing  the  gelatine,  etc.  (Fig.  108). 


1 66  CULTURE    MEDIA 

5.  Keep  the  water  in  the  steam  can  vigourously  boil- 
ing,  and  so  steam  at  100°  C.,  bubbling  through  the 
medium  mass,  for  ten  minutes,  by  which  time  complete 
solution  of  the  gelatine  is  effected.     A  certain  amount 
of  steam  will  condense  as  water  in  the  medium  flask 
during  this  process — hence  the  necessity  for  the  use 
of  double  strength  meat   extract — but  if   the   water 
bath  is  kept  boiling  this  condensation  will  not  exceed 
100  c.c. 

6.  Weigh  the  flask  and  its  contents;  then   (ins1 
grammes  -f  weight  of  the  flask)  minus  (weight  of  the 
flask  and  its  contents)  equals  the  weight  of  water  re- 
quired to  make  up  the  bulk  to  i  litre.     The  addition  of 
the  requisite  quantity  of  water  is  carried  out  as  follows : 

In  one  pan  of  the  trip  balance  place  the  counterpoise 
of  the  tared  flask  (or  its  equivalent  in  weights)  to- 
gether with  the  weights  making  up  the  calculated 
medium  weight.  In  the  opposite  pan  place  the  flask 
containing  the  medium  mass.  Now  add  boiling 
distilled  water  from  a  wash  bottle  until  the  two  pans 
are  exactly  balanced. 

7.  Titrate  and  estimate  the  reaction  of  the  medium 
mass;  control  the  result.     Calculate  the  amount   of 
soda  solution  required  to  make  the  reaction  of  the 
medium  mass   +10  (i.  e.,  calculate  for  1000  c.c.,  less 
the  quantity  used  for  the  titrations) . 

8.  Add  the  necessary  amount  of  soda  solution  and 
heat  in  the  steamer  at  100°  C.  for  twenty  minutes,  to 
precipitate  the  phosphates,  etc. 

9.  Allow  the  medium  mass  to  cool  to  60°  C.     Well 
whip  the  whites  of  two  eggs,  add  to  the  contents  of  the 
flask  and  replace  in  the  steamer  at  100°  C.  for  about 
half  an  hour  (until  the  egg-albumen  has  coagulated 

1  This  figure  is  obtained  by  adding  together  i  litre  water,  1000  grammes; 
10  per  cent,  gelatine,  100  grammes;  i  per  cent,  peptone,  10  grammes;  0.5 
per  cent,  salt,  5  grammes;  total,  1115  grammes.  Modifications  of  the  above 
process,  as  to  quantities  and  percentages,  will  require  corresponding  altera- 
tions of  the  figures.  The  average  weight  of  a  measured  litre  of  10  per  cent, 
nutrient  gelatine  when  prepared  in  this  way  after  filter  at  ion  is  1080  grammes. 


NUTRIENT  AGAR-AGAR  167 

and  formed  large,  firm  masses  floating  on  and  in  clear 
gelatine) . 

10.  Filter  through  papier  Chardin  into  a  sterile  flask. 

11.  Tube  in  quantities  of  10  c.c. 

12.  Sterilise  in  the  steamer  at  100°  C.  for  twenty 
minutes  on  each  of  three  consecutive  days — i.  e.,  by 
the  discontinuous  method. 

Nutrient  Agar=agar. — 

1 .  Weigh  a  2 -litre  flask  and  note  the  weight — or  coun- 
terpoise exactly. 

2 .  Measure  out  double  strength  meat  extract,  500  c.c., 
into  the  " tared"  flask. 

3.  Weigh  out  and  mix  10  grammes  of  peptone,   5 
grammes  of  salt,  and  20  grammes  of  powdered  agar, 
and  make  into  a  thick  paste  with  150  c.c.  distilled 
water,  and  add  to  the  meat  extract  in  the  flask;  place 
the  flask  in  a  water-bath. 

4.  Arrange  the  steam  can  and  water-bath  as  already 
directed  (for  the  preparation  of  gelatine)  and  figured. 

5.  Bubble   live    steam    (at    100°    C.)  through    the 
medium  mass,  for  twenty-five  minutes,  by  which  time 
complete  solution  of  the  agar  is  effected. 

6.  Now  weigh  the  flask  and  its  contents;  then  (I0351 
grammes  +  weight  of  flask)  minus  (weight  of  flask  and 
its  contents)  equals  the  weight  of  water  required  to 
make  up  the  bulk  of  the  medium  to  i  litre.     Add  the 
requisite  amount   (see  preparation   of  gelatine,  page 
1 66,  step  6). 

7.  Titrate,  and  estimate  the  reaction  of  the  medium 
mass;  control  the  result.     Calculate  the  amount  of 

1  This  figure  is  obtained  by  adding  together  i  litre  of  water  (meat  ex- 
tract), 1000  grammes;  2  per  cent,  agar,  20  grammes;  i  per  cent,  peptone, 
10  grammes;  0.5  per  cent,  salt,  5  grammes — total  1035  grammes.  Modi- 
fications of  the  process  as  to  quantities  or  percentages  will  nessitate  corres- 
ponding alterations  in  the  calculated  medium  figure.  The  average  weight 
of  a  measured  litre  of  2  per  cent,  agar  when  prepared  in  this  way,  after 
filtration,  is  1010.5  grammes. 


1 68  CULTURE   MEDIA 

soda  solution  required  to  make  the  reaction  of  the 
medium  mass  +  10  (i.  e.,  calculated  for  1000  c.c.,  less 
the  quantity  used  for  the  titrations). 

8.  Add  the  necessary  amount  of  soda  solution  and 
replace  in  the  steamer  for  twenty  minutes  (to  complete 
the  precipitation  of  the  phosphates,  etc.) . 

9.  Allow  the  medium  mass  to  cool  to  60°  C.     Well 
whip  the  whites  of  two  eggs,  add  to  the  contents  of 
the  flask,  and  replace  in  the  steamer  at  100°  C.  for 
about  one  hour  (until  the  egg-albumen  has  coagulated 
and  formed  large,  firm  masses  floating  on  and  in  clear 
agar.) 

10.  Filter  through  papier  Char  din,  by  the  aid  of  a 
hot-water  funnel,  if  necessary  (Fig.  101),  into  a  sterile 
flask. 

11.  Tube  in  quantities  of  10  c.c.  or  15  c.c. 

12.  Sterilise  in  the   steamer  at  100°  C.  for  thirty 
minutes  on  each  of  three  consecutive  days — i.  e.,  by  the 
discontinuous  method. 

Blood=serum  (Inspissated). — 

1.  Sterilise  cylindrical  glass  jar  (Fig.  109)  and  its 
cover  by  dry  heat,  or  by  washing  first  with  ether  and 
then  with  alcohol  and  drying. 

2.  Collect  blood  at  the  slaughter  house  from  ox  or 
sheep  in  the  sterile  cylinder. 

3.  Allow  the  vessel  to  stand  for  fifteen  minutes  for 
the  blood  to  coagulate.     (This  must  be  done  before 
leaving  the  slaughter-house,  otherwise  the  serum  will 
be  stained  with  haemoglobin.) 

4.  Separate  the  clot  from  the  sides  of  the  vessel  by 
means  of  a  sterile  glass  rod  (the  yield  of  serum  is  much 
smaller  when  this  is  not  done),  and  place  the  cylinder 
in  the  ice-chest  for  twenty-four  hours. 

5.  Remove  the  serum  with  sterile  pipettes,  or  syphon 
it  off,  and  fill  into  sterile  tubes   (5  c.c.  in  each)  or 
flasks. 


BLOOD-SERUM 


l6o 


6.  Heat  tubes  containing  serum  to  56°  C.  in  a  water- 
bath  for  half  an  hour  on  each  of  two  successive  days. 

7.  On  the  third  day,  heat  the  tubes,  in  a  sloping 
position,  in  a  serum  inspissator  to  about  72°  C.     (A 
coagulum  is  formed  at  this  temperature  which  is  fairly 
transparent;  above  72°  C.,  a  thick  turbid  coagulum  is 
formed.) 


FIG.  109. — Blood-serum  jar  with  wicker  basket  for  transport. 

The  serum  inspissator  (Fig.  no)  in  its  simplest  form 
is  a  double-walled  rectangular  copper  box,  closed  in  by 
a  loose  glass  lid,  and  cased  in  felt  or  asbestos — the 
space  between  the  walls  is  filled  with  water.  The 
inspissator  is  supported  on  adjustable  legs  so  that  the 
serum  may  be  solidified  at  any  desired  "slant,"  and 
is  heated  from  below  by  a  Bunsen  burner  controlled  by 
a  thermoregulator.  The  more  elaborate  forms  resemble 
the  hot-air  oven  (Fig.  26)  in  shape  and  are  provided 
with  adjustable  shelves  so  that  any  desired  obliquity 
of  the  serum  slope  can  be  obtained. 

8.  Place  the  tubes  in  the  incubator  at  37°  C.  for 


1 70  CULTURE    MEDIA 

forty-eight  hours  in  order  to  eliminate  those  that  have 
been  contaminated.  Store  the  remainder  in  a  cool 
place  for  future  use. 

Alternative  Method. 

Steps  1-5  as  above. 

6.  Sterilise  the  serum  by  the  fractional  method — 
that  is,  by  exposure  in  a  water-bath  to  a  temperature 
of  56°  C.  for  half  an  hour  on  each  of  six  consecutive 
days ;  store  in  the  fluid  condition. 

7.  Coagulate  in  the  inspissator  when  needed. 


FIG.  no. — Serum  inspissator. 
Serum  Water. — 

This  forms  the  basis  of  many  useful  media,  and  is  prepared  as 
follows : 

1.  Collect  blood  in  the  slaughterhouse  (see  page  168)  and  when 
firmly  clotted  collect  all  the  expressed  serum  and  measure  in  a 
graduated  cylinder. 

2.  For  every  100  c.c.  of  serum  add  300  c.c.  distilled  water  and 
mix  in  a  flask. 

3.  Heat  the  mixture  in  the  steamer  at   100°  C.  for  thirty  min- 
utes.     (This  destroys  any  diastatic  ferment  present  in  the  serum 
and  partially  sterilises  the  fluid.) 

4.  Filter  if  turbid. 

5.  If  not  needed  at  once  complete  the  sterilisation  of  the  serum 
water  by  two  subsequent  steamings  at  100°  C.  for  twenty  minutes 
at  twenty-four  hour  intervals. 


CITRATED    BLOOD   AGAR  171 

Citrated  Blood  Agar.    Guy's. — 

1.  Kill  a  small  rabbit  with  chloroform  vapour,  and 
nail  it  out  on  a  board  (as  for  a  necropsy) ;  moisten  the 
hair  thoroughly  with  2  per  cent,  solution  of  lysol. 

2.  Sterilise  several  pairs  of  forceps,  scissors,  etc.  by 
boiling. 

3.  Reflect   the   skin   over  the  thorax  with  sterile 
instruments. 

4.  Open  the  thoracic  cavity  by  the  aid  of  a  fresh 
set  of  sterile  instruments. 

5.  Open  the  pericardium  with  another  set  of  sterile 
instruments. 

6.  Sear  the  surface  of  the  left  ventricle  with  a  red- 
hot  iron. 

7.  Take  a  steiile  capillary  pipette  (Fig.  13,^);  break 
off  the  sealed  extremity  with  a  pair  of  sterile  forceps. 

8.  Steady  the  heart  in  a  pair  of  forceps  and  thrust 
the  point  of  the  pipette  through  the  wall  of  the  ven- 
tricle and  through   the  seared  area,  apply  suction  to 
the  plugged  end  of  the  pipette  and  fill  it  with  blood. 

9.  Transfer  the  entire  quantity  of  blood  collected 
from  the  rabbit's  heart  to  a  small  Erlenmeyer  flask 
containing  a  number  of  sterile  glass  beads  and  5  c.c. 
concentrated  sod.  citrate  solution.     (Seepage  378.) 

10.  Agitate  thoroughly  and  set  aside  for  a  couple  of 
hours. 

1 1 .  Melt  up  several  tubes  of  nutrient  agar  (see  page 
167)  and  cool  to  42°  C. 

12.  With  a  sterile  10  c.c.  graduated  pipette  transfer 
i  c.c.  citrated  blood  from  the  Erlenmeyer  flask  to  each 
tube  of  liquefied  agar.     Rotate  the  tube  between  the 
hands  in  order  to  diffuse  the  citrated   blood  evenly 
throughout  the  agar. 

13 .  Place  the  tubes  in  a  sloping  position  and  allow  the 
medium  to  set. 

14.  Place  tubes  of  blood  agar  for  forty-eight  hours  in 


172  CULTURE   MEDIA 

the  incubator  at  37°  C.  and  at  the  end  of  that  time 
eliminate  any  contaminated  tubes. 

15.  Store  such  tubes  as  remain  sterile  for  future  use. 

Milk.- 

1.  Pour  i  litre  of  fresh  cow's  or  goat's  milk  into  a 
large  separating  funnel,  and   heat  in  the  steamer  at 
100°  C.  for  one  hour. 

2.  Remove  from  the  steamer  and  estimate  the  re- 
action of  the  milk  (normal  cows'  milk  averages  +17). 
If  of  higher  acidity  than  +20,  or  lower  than  H-io,  re- 
ject this   sample  of  milk  and  proceed  with  another 
supply  of  milk  from  a  different  source. 

Reject  milk  to  which  antiseptics  have  been  added  as 
preservatives. 

3.  Allow  the  milk  to  cool,  when  the  fat  or  cream 
will  rise  to  the  surface  and  form  a  thick  layer. 

4.  Draw  off  the  subnatant  fat-free  milk  into  sterile 
tubes  (10  c.c.  in  each). 

5.  Sterilise  in  the  steamer  at   100°  C.  for  twenty 
minutes  on  each  of  five  successive  days. 

6.  Incubate   at   37°   C.    for   forty-eight   hours   and 
eliminate    any    contaminated    tubes.     Store    the    re- 
mainder for  future  use. 

Litmus  Milk. — 

1.  Prepare  milk  as  described  above,  sections  i  to  3. 

2.  Draw  off  the  subnatant  fat-free  milk  into  a  flask. 

3.  Add  sterile  litmus  solution,  sufficient  to  colour 
the  milk  a  deep  lavender. 

4.  Tube,  sterilise,  etc.,  as  for  milk. 

Nutrose  Agar  (Eyre). — 

(This  is  a  modification  of  the  well  known  Drigalski- 
Conradi  medium  originally  introduced  for  the  isolation 
of  B.  typhosus). 

i.  Collect   250  c.c.  perfectly  fresh  ox  serum   (vide 


NUTROSE   AGAR  173 

Blood  Serum,  page  168,  steps  i  to  5)  and  add  to  it 
450  c.c.  sterile  distilled  water. 

2.  Weigh  out  agar  powder,  20  grammes,  and  emulsify 
it  with  250  c.c.  of  the  cold  serum  water. 

3.  Weigh  out 

Witte's  peptone 10  grammes 

Sodium  chloride 5  grammes 

Nutrose 10  grammes 

and  dissolve  in  200  c.c.  of  serum  water  heated  to  80°  C. 

4.  Mix  the  agar  emulsion  and  the  peptone-nutrose 
solution  in  a  "  tared  "  flask  of  2 -litre  capacity  and  add  a 
further  100  c.c.  serum  water. 

5.  Complete  the  solution  of  the  various  ingredients 
by  bubbling  live  steam  through  the  flask  as  in  making 
nutrient  agar. 

6.  Add  further  250  c.c.  serum  water. 

7.  Weigh   the  flask  and   its   contents:   then   (1045 
grammes  +  weight  of  flask)  minus  (weight  of  flask  and 
its  present  contents)  =  weight  of  fluid  required  to  make 
up  the  bulk  of  the  medium  to  i  litre.     Add  the  requisite 
amount  of  sterile  distilled  water. 

8.  Titrate  and  estimate  the  reaction  of  the  medium 
mass.     Then  standardise  to  reaction  of  +2.5. 

9.  Clarify  with  egg,  and  filter  as  for  nutrient  agar. 
(In    clarifying,  after   the   addition   of   the  egg  white 
the  mixture  should   be  in   the  steamer  for  full   two 
hours.) 

10.  After  filtration  is  complete  measure  the  filtrate, 
and  to  every  150  c.c.  of  the  medium  add: 

Litmus  solution  (Kahlbaum) 20  c.c. 

Krystal  violet  aqueous  solu- 
tion (i :  1000)  (B.  Hoechst) i .  5  c.c. 

Lactose ....1.5  grammes 

11.  Tube  in  quantities  of  15  c.c. 

12.  Sterilise  in  the  steamer  at  100°  C.  for  thirty  min- 
utes on  each  of  three  successive  days — i.e.,  by  the  dis- 
continuous method  for  three  days. 


174 


CULTURE    MEDIA 


Egg  Medium  (Dorset). — 

1.  Prepare  1000  c.c.  of  a  0.85  per  cent,  solution  of 
sodium  chloride  in  a  stout  2 -litre  flask. 

2.  Sterilise  in  the  autoclave  at  120°  C.  for  twenty 
minutes.     Cool  to  20°  C. 

3.  Take    12   fresh  eggs;  wash  the  shells  first  with 
water  then  with  undiluted  formalin:  allow  the  shells 
to  dry. 

4.  Break  the  eggs  into  a  sterile  graduated  cylinder 
and  measure  the  total  volume  of  the  mixed  whites 
and  yolks.     Add  one  part  sterile  saline  solution  to  three 
parts  mixed  eggs. 

5.  Transfer  this  mixture  to  a  large  wide-mouthed 
stoppered  bottle  previously  sterilised.     Add  sterile  glass 
beads  and  shake  thoroughly  in  a  mechanical  shaker  for 
about  thirty  minutes,  or  whip  with  an  egg- whisk. 

6.  Filter  through  coarse  butter  muslin  into  a  sterile 
flask. 

NOTE. — A  few  drops  of  alcoholic  solution  of  basic  fuchsin  (suffi- 
cient to  give  a  definite  pink  colour) ,  or  a  few  drops  of  waterproof 
Chinese  ink  added  to  the  medium  at  this  stage  facilitates  the  sub- 
sequent "fishing"  of  colonies. 

7.  Tube  in  quantities  of  10  c.c. 

8.  Solidify  in  the  sloping  position  in  the  inspissator 
at  75°  C.  for  one  hour. 

9.  Place  the  tubes  for  forty-eight  hours  in  the  incu- 
bator at  37°  C.,  and  eliminate  any  contaminated  tubes. 

To  prevent  drying,  0.5  c.c.  glycerine  bouillon  (see 
page  209)  may  be  added  to  each  tube  between  steps  8 
and  9. 

10.  Cap  those  tubes  of  media  which  remain  sterile 
with  india-rubber  caps  and  store  for  future  use. 

Potato.— 

i.  Choose  fairly  large  potatoes,  wash  them  well,  and 
scrub  the  peel  with  a  stiff  nail-brush. 


BEER    WORT 


175 


2.  Peel  and  take  out  the  eyes. 

3.  Remove  cylinders  from  the  longest  diameter  of 
each  potato  by  means  of  an  apple-corer  or  a  large  cork- 
borer  (i.  e.,  one  of  about  1.4  cm.  diameter). 

The  reaction  of  the  fresh  potato  is  strongly  acid  to 
phenolphthalein.  If,  therefore,  the  potatoes  are  re- 
quired to  approximate  +10,  as  for  the  cultivation  of 
some  of  the  vibrios,  the  cylinders  should 
be  soaked  in  a  i  per  cent,  solution  of 
sodium  carbonate  for  thirty  minutes. 

4.  Cut  each  cylinder  obliquely  from  end 
to   end,  forming  two  wedge-shaped  por- 
tions. 

5 .  Place  a  small  piece  of  sterilised  cot- 
ton-wool, moistened  with  sterile  water,  at 
the  bottom  of  a  sterile  test-tube;  insert 
the  potato  wedge  into  the  tube  so  that 
its  base  rests  upon  the  cotton- wool.     Now 
plug  the  tube  with  cotton-wool  (Fig.  in). 

6.  Sterilise  in  the  steamer  at  100°  C.  for 
twenty  minutes  on  each  of  five  consecutive 
days. 

NOTE. — The  cork  borer  reserved  for  cutting  the 
potato  cylinders  should  be  silver  electro- plated  both 
inside  and  out,  and  the  knife  used  for  dividing  the 
cylinders  should  be  of  silver  or  silver  plated.  When 
these  precautions  are  adopted  the  potato  wedges 
will  retain  their  white  color  and  will  not  show  the  discoloration 
so  often  observed  when  steel  instruments  are  employed. 

Beer  Wort. — Wort  is  chiefly  used  as  a  medium  for 
the  cultivation  of  yeasts,  moulds,  etc.,  both  in  its 
fluid  form  and  also  when  made  solid  by  the  addition  of 
gelatine  or  agar.  The  wort  is  prepared  as  follows : 

1.  Weigh  out  250  grammes  crushed  malt  and  place 
in  a  2 -litre  flask. 

2.  Add  1000  c.c.  distilled  water,  heated  to  70°  C., 
and  close  the  flask  with  a  rubber  stopper. 


FIG.  in. — 
Potato  tube. 


176  CULTURE   MEDIA 

3.  Place  the  flask  in  a  water-bath  regulated  to  60°  C. 
and  allow  the  maceration  to  continue  for  one  hour. 

4.  Strain  through  butter  muslin  into  a  clean  flask 
and  heat  in  the  steamer  for  thirty  minutes. 

5.  Filter  through  Swedish  filter  paper. 

6.  Tube  in  quantities  of  10  c.c.  or  store  in  flasks. 

7.  Sterilise  in  the  steamer  at   100°  C.  for  twenty 
minutes  on  each  of  three  consecutive  days. 

The  natural  reaction  of  the  wort  should  not  be  inter- 
fered with. 

NOTE. — It  is  sometimes  more  convenient  to  obtain  "unhopped"1 
beer  wort  direct  from  the  brewery.  In  this  case  it  is  diluted 
with  an  equal  quantity  of  distilled  water,  steamed  for  an  hour, 
filtered,  filled  into  sterile  flasks  or  tubes,  and  sterilised  by  the 
discontinuous  method. 

Wort  Gelatine.— 

1.  Measure  out  wort  (prepared  as  above),  900  c.c., 
into  a  sterile  flask. 

2.  Weigh  out  gelatine,  100  grammes  (  =  10  per  cent.), 
and  add  it  to  the  wort  in  the  flask. 

3.  Bubble  live  steam  through  the  mixture  for  ten 
minutes,  to  dissolve  the  gelatine. 

4.  Cool  to  60°  C. ;  clarify  with  egg  as  for  nutrient 
gelatine  (vide  page  164). 

5.  Filter  through  papier  Chardin. 

6.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

Wort  Agar.— 

1.  Measure  out  wort   (as  above),   700  c.c.,   into  a 
sterile  flask. 

2.  Weigh  out  powdered  agar,  20  grammes;  mix  into 
a  smooth  paste  with  200  c.c.  of  cold  wort  and  add  to 
the  wort  in  the  flask. 

3 .  Bubble  live  steam  through  the  mixture  for  twenty 
minutes,  to  dissolve  the  agar. 

1  "  Hopped"  wort  exerts  a  toxic  effect  upon  many  bacteria,  including  the 
lactic  acid  bacteria. 


MEDIA  177 

4.  Cool  to  60°  C. ;  clarify  with  egg  as  for  nutrient 
agar  (vide  page  167). 

5.  Filter  through  papier   Chardin,    using    the    hot- 
water  funnel. 

6.  Tube,  and  sterilise  as  for  nutrient  agar. 

Peptone  Water  (Dunham). — 

1.  Weigh   out   Witte's   peptone,   10    grammes,  and 
salt,  5  grammes,  and  emulsify  with  about  250  c.c.  of 
distilled  water  previously  heated  to  60°  C. 

2.  Pour  the  emulsion  into  a  litre  flask  and  make  up 
to  1000  c.c.  by  the  addition  of  distilled  water. 

3.  Heat  in  the  steamer  at  100°  C.  for  thirty  minutes. 

4.  Filter  through  Swedish  filter  paper. 

5.  Tube  in  quantities  of  10  c.c.  each. 

6.  Sterilise  in  the  steamer  at   100°  C.  for  twenty 
minutes  on  each  of  three  consecutive  days. 

"Sugar"  or  "Carbohydrate"  Media. — 

Formerly  the  ability  of  bacteria  to  induce  hydrolytic 
changes  in  carbohydrate  substances  was  observed  only 
in  connection  with  a  few  well-defined  sugars,  but  of 
recent  years  it  has  been  shown  that  when  using 
litmus  as  an  indicator  these  so-called  "  fermentation 
reactions"  facilitate  the  differentiation  of  closely 
allied  species,  and  the  list  of  substances  employed 
in  this  connection  has  been  considerably  extended. 
The  media  prepared  with  them  are  now  no  longer 
regarded  as  special,  but  are  comprised  in  the  "  stock 
media"  of  the  laboratory.  The  chief  of  these  sub- 
stances are  the  following,  arranged  in  accordance  with 
their  chemical  constitution : 

Monosaccharides Dextrose  (glucose),  laevulose,  galactose, „ . 

mannose,  arabinose,  xylose. 

Disaccharides Maltose,  lactose,  saccharose. 

Trisaccharides Raffinose  (meltitose) . 

Polysaccharides Dextrin,  inulin,  starch,  glycogen,  amidon. 

Glucosides Amygdalin,    coniferin,    salicin,    helicin, 

phlorrhizin. 

12 


178  CULTURE   MEDIA 

Polyatomic  alcohols Trihydric,  Glycerin. 

Tetrahydric,  Erythrite. 

Pentahydric,  Adonite 

Hexahydric,  Dulcite,  (dulcitol  or  mel- 
ampirite) ,  isodulcite  (rhamnose) ,  man- 
nite  (mannitol),  sorbite  (sorbitol), 
inosite. 

These  substances  should  be  obtained  from  Kahlbaum 
(of  Berlin);  in  the  pure  form,  and  when  possible 
as  large  crystals,  and  the  method  of  preparing  a 
medium  containing  either  of  them  may  be  exem- 
plified by  describing  Dextrose  Solution. 

Dextrose  Solution. — 

1.  Weigh  out 

Peptone 20  grammes 

Glucose     . i  o  grammes 

and  grind  together  in  a  mortar;  then  emulsify  in  100  c.c. 
of  distilled  water  heated  to  60°  C. 

2.  Place  in  a  flask  and  add 

Distilled  water 850  c.c. 

3.  Steam   in   the   steamer   at    100°    C.  for   twenty 
minutes  to  dissolve  the  peptone  and  glucose. 

4.  Add 

Kubel-Tiemann  litmus  solution  (Kahlbaum)       ...    50  c.c. 

(The  substances  enumerated  above  react  acid  to 
phenolphthalein,  but  variously  toward  the  neutral 
litmus  solution.  To  such  as  react  acid,  add  very 
cautiously  °  sodium  hydrate  solution  to  the  medium 
in  bulk  until  the  neutral  tint  has  returned) . 

5.  Fill  into  tubes  in  which  have  previously  been 
placed  the  inverted  Durham's  gas  tubes. 

6.  Sterilise  in   the  steamer  at    100°   C.  for   twenty 
minutes  on  each  of  three  successive  days. 

NOTE. — On  no  account  should  these  media  be  sterilised  in  the 
autoclave,  as  temperatures  above  100°  C.  themselves  induce 
hydrolytic  changes  in  the  substances  in  question.  It  is  equally 


NEUTRAL    LITMUS    SOLUTION  179 

important  that  the  twenty  minutes  should  not  be  exceeded  in 
sterilisation,  as  neglect  of  this  precaution  may  discolour  the 
litmus  or  lead  to  the  production  of  yellowish  tints  when  the 
tubes  are  subsequently  inoculated  with  acid-forming  bacteria. 

Neutral  Litmus  Solution. 

The  most  satisfactory  is  the  Kubel-Tiemann,  pre- 
pared by  Kahlbaum.  It  can  however  be  made  in  the 
laboratory  as  follows : 

1.  Weigh  out 

Commercial  litmus 50  grammes, 

and  place  in  a  well  stoppered  500  c.c.  bottle;  measure 
out  and  add  300  c.c.  alcohol  95  per  cent. 

2.  Shake  well  at  least  once  a  day  for  seven  days — 
the  alcohol  acquires  a  green  colour. 

3.  Decant  off  the  green  alcohol  and  fill  a  further  300 
c.c.  95  per  cent,  alcohol  into  the  bottle  and  repeat  the 
shaking. 

4.  Repeat  this  process  until  on  adding  fresh  alcohol 
the  fluid  only  becomes  tinged  with  violet. 

5.  Pour  off  the  alcohol,  leaving  the  litmus  as  dry  as 
possible.     Connect  up  the  bottle  to  an  air  pump  and 
evaporate  off  the  last  traces  of  alcohol. 

6.  Transfer  the  dry  litmus  to  a  litre  flask,  measure  in 
600  c.c.  distilled  water  and  allow  to  remain  in  contact 
24  hours  with  frequent  shakings. 

7.  Filter  the  solution  into  a  clean  flask  and  add  one  or 
two  drops  of  pure  concentrated  sulphuric  acid  until  the 
litmus  solution  is  distinctly  wine-red  in  colour. 

8.  Add  excess  of  pure  solid  baryta  and  allow  to  stand 
until  the  reaction  is  again  alkaline. 

9.  Filter. 

10.  Bubble  CO2  through  the  solution  until  reaction 
is  definitely  acid. 

11.  Sterilise  in  the  steamer  at   100°  C.  for  thirty 
minutes  on   each    of    three    consecutive    days.     This 
sterilises  the  solution  and  also  drives  off  the  carbon 
dioxide,  leaving  the  solution  neutral. 


l8o  CULTURE    MEDIA 

Media  for  anaerobic  cultures.  In  addition  to  the 
foregoing  media,  all  of  which  can  be,  and  are  employed 
in  the  cultivation  of  anaerobic  bacteria,  certain  special 
media  containing  readily  oxidised  substances  are  com- 
monly used  for  this  purpose.  The  principal  of  these 
are  as  follows : 

Bile  Salt  Broth   (MacConkey).— 

1.  Weigh  out  Witte's  peptone,    20  grammes   (  =  2   per  cent.), 
and  emulsify  with  200  c.c.  distilled  water  previously  warmed  to 
60°  C. 

2.  Weigh  out  sodium   taurocholate   (commercial),    5    grammes 
(  =  0.5   per  cent.),   and  glucose,    5   grammes   (  =  0.5   per  cent.), 
and  dissolve  in  the  peptone  emulsion. 

3.  Wash  the  peptone  emulsion  into  a  flask  with  800  c.c.  distilled 
water,  and  heat  in  the  steamer  at  100°  C.  for  twenty  minutes. 

4.  Filter  through  Swedish  filter  paper  into  a  sterile  flask. 

5.  Add  sterile  litmus  solution  sufficient  to  colour  the  medium 
to  a  deep  purple,  usually  13  per  cent,  required. 

6.  Fill,    in    quantities  of    10   c.c.,  into   tubes   containing  small 
gas  tubes  (vide  Fig.    104,  page  161).     Sterilise  in  the  steamer  at 
1 00°  C.  for  twenty  minutes  on  each  of  three  consecutive  days. 

Glucose  Formate  Bouillon   (Kitasato). — 

1.  Measure   out   nutrient  bouillon,    1000    c.c.    (vide   page    163, 
sections  i  to  6). 

2.  Weigh  out  glucose,    20  grammes   (  =  2   per  cent.),    sodium 
formate,  4  grammes  (  =  0.4  per  cent.) ,  and  dissolve  in  the  fluid. 

3.  Tube,  and  sterilise  as  for  bouillon. 

Glucose  Formate  Gelatine  (Kitasato). — 

1.  Prepare  nutrient  gelatine  (vide  page  164,  sections  i  to  7)  and 
measure  out  1000  c.c. 

2.  Weigh  out  glucose,  20  grammes  (  =  2  per  cent.),  and  sodium 
formate,  4  grammes  (  =  0.4  per  cent.),  and  dissolve  in  the  hot 
gelatine. 

3.  Filter  through  papier  Chardin. 

4.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

Glucose  Formate  Agar  (Kitasato). — 

1.  Prepare  nutrient    agar    (vide    page    167,  sections    i    to    8). 
Measure  out  1000  c.c. 

2.  Weigh  out  glucose,    20  grammes    (  =  2   per  cent.),   sodium 
formate,  4  grammes  (  =  0.4  per  cent.),  and  dissolve  in  the  agar. 

3.  Tube,  and  sterilise  as  for  nutrient  agar. 


SULPHINDIGOTATE   A  GAR  l8l 

Sulphindigotate  Bouillon   (Weyl).— 

1.  Measure   out  nutrient  bouillon    (vide  page    163,   sections    i 
to  6  1000  c.c.). 

2.  Weigh   out   glucose,    20   grammes    (  =  2    per  cent.),    sodium 
sulphindigotate,    i    gramme    ( =  o .  i    per  cent.) ,    and   dissolve  in 
the  fluid. 

3.  Tube,  and  sterilise  as  for  bouillon. 

Sulphindigotate  Gelatine  (Weyl).— 

1.  Prepare   nutrient  gelatine  (vide  page  164,  sections  i  to   7). 
Measure  out  1000  c.c. 

2.  Weigh  out  glucose,  20  grammes  (  =  2  per  cent.),  and  sodium 
sulphindigotate,    i    gramme    ( =  o .  i    per   cent.) ,    and   dissolve  in 
the  hot  gelatine. 

3.  Filter  through  papier  Chardin. 

4.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

Sulphindigotate  Agar. — 

1.  Prepare   nutrient    agar    (vide   page    167,    sections    i    to    8). 
Measure  out  1000  c.c. 

2.  Weigh   out   glucose,    20   grammes    (  =  2    per   cent.),    sodium 
sulphindigotate,    i    gramme    (  =  0.1    per   cent.),    and   dissolve  in 
the  hot  agar. 

3.  Tube,  and  sterilise  as  for  nutrient  agar. 

NOTE. — The  Sulphindigotate  media  are  of  a  blue  colour,  which 
during  the  growth  of  anaerobic  bacteria  is  oxidised  and  decolour- 
ised to  a  light  yellow. 


XII.  SPECIAL  MEDIA. 

In  this  chapter  are  collected  a  number  of  media 
which  have  been  elaborated  by  various  workers  for 
special  purposes,  grouped  together  under  headings 
which  indicate  their  chief  utility.  In  many  instances 
the  name  of  the  originator  of  the  medium  is  given,  but 
without  reference  to  his  original  instructions,  since 
these  are  in  many  cases  inadequate  to  the  require- 
ments of  the  isolated  worker,  who  would  probably  fail 
to  reproduce  the  medium  in  a  form  giving  the  results 
attributed  to  it  by  its  author.  Such  modifications 
have  therefore  been  introduced  as  make  for  uniformity 
between  the  different  batches  of  media. 

A  considerable  number  of  coloured  media,  chiefly  in- 
tended for  work  with  intestinal  bacteria,  have  been 
included ;  but  beyond  the  fact  that  the  author's  modi- 
fication of  the  Drigalski-Conradi  medium  has  been  in- 
cluded amongst  the  routine  media  of  the  laboratory, 
no  comment  has  been  made  upon  their  relative  values, 
since  only  by  observation  and  practice  can  the  skill 
necessary  to  utilise  their  full  value  be  acquired. 

The  instructions  as  to  sterilisation  are  raiely  given 
in  full;  the  routine  method  of  exposure  in  the  steam 
steriliser  at  100°  C.  (without  pressure)  for  twenty  min- 
utes on  each  of  three  successive  days  for  all  fluid 
media,  and  thirty  minutes  on  each  of  three  successive 
days  for  all  liquefiable  or  solid  media  must  be  carried 
out ;  and  only  when  these  general  rules  are  to  be  de- 
parted from  are  further  details  given. 

182 


INOSITE-FREE    MEDIA,  183 

Media  for  the  Study  of  the  Chemical  Composition  of  Bacteria. 
Asparagin  Medium  (Uschinsky). — 

1.  Weigh  out  and  mix 

Asparagin 3 . 4  grammes 

Ammonium  lactate 10.0  grammes 

Sodium  chloride 5.0  grammes 

Magnesium  sulphate      0.2  gramme 

Calcium  chloride o .  i  gramme 

Acid  potassium  phosphate  (KH2PO4)  i .  o  gramme 

2.  Dissolve  the  mixture  in  distilled  water  1000  c.c. 

3.  Add  glycerine,  40  c.c. 

4.  Tube,  and  sterilise  as  for  nutrient  bouillon. 

Asparagin  Medium  (Frankel  and  Voges). — 

1.  Weigh  out  and  mix 

Asparagin 4  grammes 

Sodium  phosphate,  (Na2HPOj  1 2OH.    .  2  grammes 

Ammonium  lactate 6  grammes 

Sodium  chloride 5  grammes 

and  dissolve  in 

Distilled  water 1000  c.c. 

2.  Tube,  and  sterilise  as  for  nutrient  bouillon. 

NOTE. — Either  of  the  above  asparagin  media,  after  the  addition 
of  10  per  cent,  gelatine  or  1.5  per  cent,  agar,  may  be  advan- 
tageously employed  in  the  solid  condition. 

Proteid  Free  Broth   (Uschinsky).— 

1.  Weigh  out  and  mix 

Calcium  chloride .    .    o .  i  gramme 

Magnesium  sulphate       0.2  gramme 

Acid  potassium  phosphate  (KH2POJ  .  2.0  grammes 
Potassium  aspartate  .    ...'....    3.0  grammes 

Sodium  chloride 5.0  grammes 

Ammonium  lactate 6.0  grammes 

2.  Dissolve  the  mixture  in  distilled  water  1000  c.c. 

3.  Add  glycerine  30  c.c. 

4.  Tube  and  sterilise  as  for  nutrient  broth. 

Media  for  the  Study  of  Bio-chemical  Reaction. 

Inosite=free  Media — Bouillon   (Durham).— 

1.  Prepare    meat    extract,     1000    c.c.    (vide    page    148),    from 
bullock's  heart  which  has  been  "hung"  for  a  couple  of  days. 

2.  Prepare  nutrient  boullion  (+10),  1000  c.c.  (vide,  page  .161), 
from  the  meat  extract,  and  store  in  i-litre  flask. 


184  SPECIAL   MEDIA 

3.  Inoculate  the  bouillon  from   a  pure  cultivation  of  the   B. 
lactis  aerogenes,  and  incubate  at  37°  C.  for  forty-eight  hours. 

4.  Heat  in  the  steamer  at  100°  C.  for  twenty  minutes  to  destroy 
the  bacilli  and  some  of  their  products. 

5.  Estimate    the    reaction    of    the    medium    and    if    necessary 
restore  to  +  10. 

6.  Inoculate  the  bouillon  from  a  pure  cultivation  of  the   B. 
coli  communis  and  incubate  at  37°  C.  for  forty-eight  hours. 

7.  Heat  in  the  steamer  at  100°  C.  for  twenty  minutes. 

Now  fill  two  fermentation  tubes  with  the  bouillon,  tint  with 
litmus  solution,  and  sterilise;  inoculate  with  B.  lactis  aerogenes. 
If  no  acid  or  gas  is  formed,  the  bouillon  is  in  a  sugar-free  con- 
dition; but  if  acid  or  gas  is  present,  again  make  the  bouillon  in 
the  flask  +10,  reinoculate  with  one  or  other  of  the  above-men- 
tioned bacteria,  and  incubate;  then  test  again.  Repeat  this  till 
neither  acid  nor  gas  appears  in  the  medium  when  used  for  the 
cultivation  of  either  of  the  bacilli  referred  to  above. 

8.  After  the  final  heating,  stand  the  flask  in  a  cool  place  and 
allow   the   growth   to   sediment.     Filter  the   supernatant   broth 
through   Swedish   filter  paper.     If  the  filtrate  is   cloudy,   filter 
through  a  porcelain  filter  candle. 

9.  Tube,  and  sterilise  as  for  bouillon. 

Bouillon  prepared  in  the  above-described  manner  will  prove 
to  be  absolutely  sugar-free;  ancl  from  it  may  be  prepared  nutrient 
sugar-free  gelatine  or  agar,  by  dissolving  in  it  the  required  per- 
centage of  gelatine  or  agar  respectively  and  completing  the  medium 
according  to  directions  given  on  pages  166  and  167.  The  most  im- 
portant application  of  inosite-free  bouillon  is  its  use  in  the  prep- 
aration of  sugar  bouillons,  whether  glucose,  maltose,  lactose,  or 
saccharose,  of  exact  percentage  composition. 

Sugar  (Dextrose)  Bouillon. — 

1.  Measure   out  nutrient  bouillon,    1000   c.c.    (vide   page    163, 
sections  i  to  6)  or  sugar-free  bouillon  (vide  svpra). 

2.  Weigh  out  glucose  (anhydrous),  20  grammes  (  =  2  per  cent.), 
and  dissolve  in  the  fluid. 

3.  Tube,  and  sterilise  as  for  bouillon. 

Ordinary  commercial  glucose  serves  the  purpose  equally  well, 
but  is  not  recommended,  as  during  the  process  of  sterilisation 
it  causes  the  medium  to  gradually  deepen  in  colour. 

NOTE. — In  certain  cases  a  corresponding  percentage  of  lactose, 
maltose,  or  saccharose  is  substituted  for  glucose. 

Sugar  Gelatine. — 

1.  Prepare  nutrient  gelatine   (vide  page    164,   sections  i  to  7). 
Measure  out  1000  c.c. 

2.  Weigh  out  glucose,  20  grammes  (  =  2  per  cent.),  and  dissolve 
in  the  hot  gelatine. 


IRON  PEPTONE  SOLUTION  185 

3.  Filter  through  papier  Chardin. 

4.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

Sugar  Agar. — 

1.  Prepare    nutrient    agar    (vide   page    167,    sections    i    to    8). 
Measure  out  1000  c.c. 

2.  Weigh  out  glucose,  20  grammes  (  =  2  per  cent.),  and  dissolve 
in  the  clear  agar. 

3.  Tube,  and  sterilise  as  for  nutrient  agar. 

NOTE. — Other  "sugar"  media  are  prepared  by  substituting  a 
corresponding  percentage  of  lactose,  maltose  (or  any  other  of  the 
substances  referred  to  under  "Sugar  Media,"  page  177)  for  the 
glucose. 

Iron  Bouillon. — 

1.  Measure   out  nutrient  bouillon,    1000   c.c.    (vide  page    141, 
sections  i  to  6). 

2.  Weigh  out  ferric  tartrate,  i  gramme  (  =  0.1  per  cent.),  and 
dissolve  it  in  the  bouillon. 

3.  Tube,  and  sterilise  as  for  bouillon. 

NOTE. — The  lactate  of  iron  may  be  substituted  for  the  tartrate. 

Lead  Bouillon. — 

1.  Measure   out  nutrient   bouillon,    1000   c.c.    (vide   page    163, 
sections  i  to  6). 

2 .  Weigh  out  lead  acetate,   i  gramme  ( =  o .  i  per  cent.) ,  and 
dissolve  it  in  the  bouillon. 

3.  Tube,  and  sterilise  as  for  bouillon. 

Nitrate  Bouillon.— 

1.  Measure   out  nutrient  bouillon,    1000   c.c.    (vide  page    163, 
sections  i  to  6). 

2.  Weigh  out  potassium  nitrate,  5  grammes  (  =  0.5  per  cent.), 
and  dissolve  it  in  the  bouillon. 

3.  Tube,  and  sterilise  as  for  bouillon. 

NOTE. — The  nitrate  of  sodium  or  ammonium  may  be  sub- 
stituted for  that  of  potassium,  or  the  salt  may  be  added  in  the 
proportion  of  from  o .  i  to  i  per  cent,  to  meet  special  requirements. 

Iron  Peptone  Solution   (Pakes).— 

1.  Weigh  out  peptone,  30  grammes,  and  emulsify  it  with  aoo 
c.c.  tap  water,  previously  heated  to  about  60°  C. 

2.  Wash  the  emulsion  into  a  litre  flask  with  800  c.c.  tap  water. 

3.  Weigh    out    salt,    5    grammes,    and    sodium    phosphate,    3 
grammes,  and  dissolve  in  the  mixture  in  the  flask. 

4.  Heat  the  mixture  in  the  steamer  at  100°  C.  for  thirty  minutes, 


1 86  SPECIAL   MEDIA 

to  complete  the  solution  of  the  peptone,  and  filter  into  a  clean 
flask. 

5.  Fill  into  tubes  in  quantities  of  10  c.c.  each. 

6.  Add  to  each  tube  o.i  c.c.  of  a  2  per  cent,  neutral  solution 
of  ferric  tartrate.      (A  yellowish-white  precipitate  forms.) 

7.  Sterilise  as  for  nutrient  bouillon. 

Lead  Peptone  Solution. — 

Prepare  as  for  iron  peptone  solution  but  in  step  6  substitute 
o.i  c.c.  of  a  i  per  Cent,  neutral  aqueous  solution  of  lead  acetate. 

Nitrate  Peptone  Solution   (Pakes).— 

1.  Weigh  out  Witte's  peptone,    10    grammes,  and  emulsify    it 
with  200  c.c.  ammonia-free  distilled  water  previously  heated  to 
60°  C. 

2.  Wash  the  emulsion  into  a  flask  and  make  up  to  1000  c.c., 
with  ammonia-free  distilled  water. 

3.  Heat  in  the  steamer  at  100°  C.  for  twenty  minutes. 

4.  Weigh  out  sodium  nitrate,   i  gramme,  and  dissolve  in  the 
contents  of  the  flask. 

5.  Filter  through  Swedish  filter  paper. 

6.  Tube,  and  sterilise  as  for  nutrient  bouillon. 

Litmus  Bouillon. — 

1.  Measure   out  nutrient  bouillon,    1000    c.c.    (vide   page    163, 
sections  i  to  6). 

2.  Add  sufficient  sterile  litmus  solution  to  tint  the  medium  a 
dark  lavender  colour.      (Media  rendered    +  10  will  usually  react 
very  faintly  alkaline  or  occasionally  neutral  to  litmus.) 

3.  Tube,  and  sterilise  as  for  bouillon. 

Rosolic  Acid  Peptone  Solution. — 

1.  Weigh  out  rosolic  acid  (corallin),  0.5  gramme,  and  dissolve 
it  in  80  per  cent,  alcohol,  100  c.c.      Keep  this  as  a  stock  solution. 

2.  Measure  out  peptone  water  (Dunham),  100  c.c.,  and  rosolic 
acid  solution,  2  c.c.,  and  mix. 

3.  Heat  in  the  steamer  at  100°  C.  for  thirty  minutes. 

4.  Filter  through  Swedish  filter  paper. 

5.  Tube,  and  sterilise  as  for  nutrient  bouillon. 

Capaldi=Proskauer  Medium,  No.  I. — 

1.  Weigh  out  and  mix 

Sodium  chloride 2.0  grammes 

Magnesium  sulphate       o.i  gramme 

Calcium  chloride 0.2  gramme 

Monopotassium  phosphate    ....2.0  grammes 

2.  Dissolve  in  water  1000  c.c.  in  a  2-litre  flask 


URINE    GELATINE  187 

3.  Weigh  out  and  mix 

Asparagin         2  grammes 

Mannite 2  grammes 

and  add  to  contents  of  flask. 

4.  Measure  out  25   c.c.  of  the  solution  and  titrate  it  against 
decinormal  sodic  hydrate,  using  litmus  as  the  indicator.     Control 
the  result  and  estimate  the  amount  of  sodic  hydrate  necessary 
to  be  added  to  render  the  remainder  of  the  solution  neutral  to 
litmus.     Add  this  quantity  of  sodic  hydrate. 

5.  Filter. 

6.  Add  litmus  solution  47.5  c.c.  (  =  5  per  cent.). 

7.  Tube,  and  sterilise  as  for  nutrient  bouillon. 

Capaldi=Proskauer  Medium     No.  II. — 

1.  Weigh  out  and  mix 

Peptone 20  grammes 

Mannite i  gramme 

2.  Dissolve  in  water  1000  c.c.  in  a  2 -litre  flask. 

3.  Neutralise  to  litmus  as  in  No.  i  (vide  supra,  Step  4). 

4.  Filter. 

5.  Add  litmus  solution  47.5  c.c.  (  =  5  per  cent.). 

6.  Tube,  and  sterilise  as  for  nutrient  bouillon. 

Urine  Media.     Bouillon. — 

1.  Collect  freshly  passed  urine  in  sterile  flask. 

2.  Place  the  flask  in  the  steamer  at  100°  C.  .for  thirty  minutes. 

3.  Filter  through  two  thicknesses  of  Swedish  filter  paper. 

4.  Tube,  and  sterilise  as  for  nutrient  bouillon. 
(Leave  the  reaction  unaltered.) 

Urine  Gelatine.— 

1.  Collect  freshly  passed  urine  in  sterile  flask. 

2.  Take  the  specific  gravity,  and,  if  above   1010,  dilute  with 
sterile  water  until  that  gravity  is  reached. 

3.  Estimate  (with  control)   at  the  boiling-point,  and  note  the 
reaction  of  the  urine. 

4.  Weigh  out  gelatine,   10  per  cent.,  and  add  to  the  urine  in 
the  flask. 

5.  Heat  in  the  steamer  at  100°  C.  for  one  hour  to  dissolve  the 
gelatine. 

6.  Estimate    the    reaction    and    add    sufficient    caustic    soda 
solution    to    restore   the    reaction    of   the    medium    mass    to    the 
equivalent  of  the  original  urine. 

7.  Cool  to  60°  C.  and  clarify  with  egg  as  for  nutrient  gelatine 
(vide  page  166). 

8.  Filter  through  papier  Chardin. 

9.  Tube,  and  sterilise  as  for  nutrient  gelatine. 


1 88  SPECIAL   MEDIA 

Urine  Gelatine  (Heller).— 

1.  Collect  freshly  passed  urine  in  sterile  flask. 

2.  Filter  through  animal     charcoal    to     remove    part    of    the 
colouring  matter. 

3.  Take  the   specific  gravity,   and  if  above   1010,  dilute  with 
sterile  water  till  this  gravity  is  reached. 

4.  Add  Witte's  peptone,  i  per  cent. ;  salt,  0.5  per  cent.;  gelatine, 
10  per  cent. 

5.  Heat  in  the  steamer  at   100°  C.   for  one  hour,  to  dissolve 
the  gelatine,  etc. 

6.  Add    normal    caustic    soda    solution    in    successive    small 
quantities,  and  test  the  reaction  from  time  to  time  with  litmus 
paper,  until  the  fluid  reacts  faintly  alkaline. 

7.  Cool  to  60°  C.  and  clarify  with  egg  as  for  nutrient  gelatine 
(vide  page  166). 

8.  Filter  through  papier  Chardin. 

9.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

Urine  Agar. — 

1.  Collect  freshly  passed  urine  in  sterile  flask. 

2.  Take  the  specific  gravity  and  if  above  1010,  dilute  with  sterile 
water  till  this  gravity  is  reached. 

3 .  Weigh  out  i .  5  per  cent,  or  2  per  cent,  powdered  agar,  and  add 
it  to  the  urine. 

4.  Heat  in  the  steamer  at  100°  C.  lor  ninety  minutes  to  dissolve 
the  agar. 

5.  Cool  to  60°  C.  and  clarify  with  egg  as  for  nutrient  agar  (vide 
page  1 68). 

6.  Filter  through  papier  Chardin,  using  the  hot-water  funnel. 

7.  Tube,  and  sterilise  as  for  nutrient  agar. 
(Leave  the  reaction  unaltered.) 

Serum  Sugar  Media  (Hiss). — 

In  these  media  the  fermentation  of  carbohydrate  substance  by 
bacterial  action  is  indicated  by  the  coagulation  of  the  serum 
proteids  in  addition  to  the  production  of  an  acid  reaction. 

Serum  Dextrose  Water  (Hiss). — 

1.  Measure  out  into  a  litre  flask 

Serum  water  (See  page  1 70) 1000  c.c. 

2.  Weigh  out 

Dextrose       10  grammes 

and  dissolve  in  the  serum  water. 

3.  Filter  through  Swedish  filter  paper. 

4.  Measure  out  and  add  to  the  medium 

Litmus  solution  (Kahlbaum)      ....    50  c.c. 


MILK   RICE  189 

5.  Tube  in  quantities  of  10  c.c.  and  sterilise  in  the  steamer  at 
100°  C.  for  twenty  minutes  on  each  of  three  successive  days. 

Laevulose,  galactose,  maltose,  lactose,  etc.,  can  be  substituted 
in  similar  amounts  for  dextrose  and  the  medium  completed  as 
above. 

Omeliansky's  Nutrient  Fluid  (For  Cellulose  Fermenters).— 

1.  Weigh  out  and  mix 

Potassium  phosphate 4.0     grammes 

Magnesium  sulphate 2.0     grammes 

Ammonium  sulphate 4.0     grammes 

Sodium  chloride 0.25   gramme 

2.  Dissolve  in  distilled  water  4000    c.c. 

3.  Flask  in  quantities  of  250  c.c. 

4.  Weigh  out  and  add  5  grammes  precipitated  chalk  to  each 
flask. 

5.  Sterilise  in  the    steamer   at  100°  C.  for  twenty  minutes  on 
each  of  three  successive  days. 


Media  for  the  Study  of  Chromogenic  Bacteria. 

Milk  Rice   (Eisenberg).— 

1.  Measure  out  nutrient  bouillon,   70  c.c.,  and  milk,   210  c.c., 
and  mix  thoroughly. 

2.  Weigh  out  rice  powder,   100  grammes,  and  rub  it  up  in  a 
mortar  with  the  milk  and  broth  mixture. 

3.  Fill  the  paste  into  sterile  capsules,  spreading  it  out  so  as 
to  form  a  layer  about  0.5  cm.  thick,  over  the  bottom  of  each. 

4.  Heat    over   a   water-bath    at    100°    C.    until    the    mixture 
solidifies. 

5.  Replace  the  lids  of  the  capsules.     Sterilise  in  the  steamer 
at  100°  C.  for  thirty  minutes  on  each  of  three  consecutive  days. 

(A  solid  medium  of  the  colour  of  cafe  au  lait  is  thus  produced.) 

Milk  Rice   (Soyka).— 

1.  Measure  out  nutrient  bouillon,   50  c.c.,  and  milk,   150  c.c., 
and  mix  thoroughly. 

2.  Weigh  out  rice  powder,    100  grammes,  and  rub  it  up  in  a 
mortar  with  the  milk  and  broth  mixture. 

3.  Fill  the  paste  into  sterile  capsules,  to  form  a  layer  over  the 
bottom  of  each. 

4.  Replace  the  lids  of  the  capsules. 

5.  Sterilise   in   the   steamer  at  100°  C.  for  thirty  minutes  on 
each  of  three  consecutive  days. 

(A  pure  white,  opaque  medium  is  thus  formed.) 


I QO  SPECIAL   MEDIA 

Media  for  the  Study  of  Phosphorescent   and   Photogenic  Bacteria. 
Fish  Bouillon.— 

1.  Weigh  out    herring,   mackerel,   or  cod,    500   grammes,   and 
place  in  a  large  porcelain  beaker  (or  enamelled  iron  pot) . 

2.  Weigh    out    sodium    chloride,     26.5    grammes;    potassium 
chloride,  0.75  gramme;  magnesium  chloride,  3.25  grammes;  and 
dissolve  in   500   c.c.   distilled  water.     Add  the   solution  to  the 
fish  in  the  beaker. 

3.  Place  the  beaker  in  a  water-bath  and  proceed  as  in  preparing 
meat  extract — i.  e.,  heat  gently  at  40°  C.  for  twenty  minutes, 
then   rapidly   raise   the   temperature   to,    and   maintain   at,    the 
boiling-point  for  ten  minutes. 

4.  Strain  the  mixture  through  butter  muslin  into  a  clean  flask. 

5.  Weigh  out  peptone,   5  grammes,  and  emulsify  with  about 
200   c.c.  of  the  hot  fish  water;  incorporate  thoroughly  with  the 
remainder  of  the  fish  water  in  the  flask. 

6.  Heat   in   the   steamer   at    100°    C.   for  twenty  minutes  to 
complete  the  solution  of  the  peptone. 

7.  Filter  through  Swedish  filter  paper. 

8.  When  the  fish  bouillon  is  cold,  if  it  is  to  be  used  as  fluid 
medium,  make  up  to  1000  c.c.  by  the  addition  of  distilled  water. 
If,  however,  it  is  to  be  used  as  the  basis  for  agar  or  gelatine  media 
store  it  in  the  "Double  Strength"  condition. 

9.  Tube  and  sterilise  as  for  nutrient  bouillon. 

As  an  alternative  method  "Marvis"  fish  food   (16  grammes) 
may  be  substituted  for  the  500  grammes  of  fresh  fish. 

Fish  Gelatine.— 

1.  Measure  out  double  strength  fish  bouillon,  500  c.c.,  into  a 
"tared"  2 -litre  flask. 

2.  Add  sheet  gelatine,  100  grammes,  cut  into  small  pieces. 

3.  Bubble  live  steam  through  the  mixture  for  fifteen  minutes  to 
dissolve  the  gelatine. 

4.  Weigh  the  flask  and  its  contents;  adjust  the  weight  to  the 
calculated  figure  for  one  litre  of  medium  (1135.5  grammes)  by  the 
addition  of  distilled  water  at  100°  C.  (vide  page  166). 

5.  Cool  to  below  60°  C.,  and  clarify  with  egg. 

6.  Filter  through  papier  Chardin. 

7.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

Shake  well  after  the  final  sterilisation,  to  aerate  the  medium. 

Fish  Gelatine -Agar. — 

1.  Weigh  out  powdered  agar,  5  grammes,  and  emulsify  it  with 
200  c.c.  double  strength  fish  bouillon. 

2.  Wash  the  emulsion  into  a  "tared"  2-litre  flask  with  300  c.c. 
fish  bouillon. 


NAEGELI'S    SOLUTION  191 

3.  Weigh  out  sheet  gelatine,    70  grammes,   cut  it  into  small 
pieces  and  add    it  to  the  contents  of  the  flask. 

4.  Bubble  live  steam  through  the  mixture  to  dissolve  the  gela- 
tine and  agar. 

5.  Weigh  the  flask  and  contents.     Adjust  the  weight  to  the 
calculated  figure  for  one  litre  of  medium  (1110.5  grammes)  by  the 
addition  of  distilled  water  at  100°  C.  (vide  page  166). 

6.  Cool  to  below  60°  C.  and  clarify  with  egg. 

7.  Filter  through  papier  Chardin. 

8.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

Shake  well  after  the  final  sterilisation,  to  aerate  the  medium. 

Media  for  the  Study  of  Yeasts  and  Moulds. 
Pasteur's  Solution. — 

(Reaction  alkaline). 

1.  Weigh   out   and  mix  the   ash  from    10   grammes  of  yeast; 
ammonium  tartrate,  10  grammes;  cane  sugar,  100  grammes. 

2.  Dissolve  the  mixture  in  distilled  water,  1000  c.c. 

3 .  Tube  or  flask,  and  sterilise  as  for  nutrient  bouillon. 

Yeast  Water  (Pasteur).— 

1.  Weigh  out  pressed  yeast,  75  grammes;  place  in  a  2-litre  flask 
and  add  1000  c.c.  distilled  water. 

2.  Heat  in  the  steamer  at  100°  C.  for  thirty  minutes. 

3.  Filter  through  papier  Chardin. 

4.  Tube  or  flask,  and  sterilise  as  for  nutrient  bouillon. 

Cohn's  Solution. — 

1.  Weigh  out  and  mix 

Acid  potassium  phosphate  (KH2PO4)  .5.0  grammes 

Calcium  phosphate 0.5  gramme 

Magnesium  sulphate       5.0  grammes 

Ammonium  tartrate       10.0  grammes 

and  dissolve  in 

Distilled  water 1000  c.c. 

2.  Tube,  or  flask  and  sterilise  as  for  nutrient  bouillon. 
Naegeli's  Solution. — 

1.  Weigh  out  and  mix 

Dibasic  potassium  phosphate  (K2HPO4)      i.o  gramme 

Magnesium  sulphate       o.-2  gramme 

Calcium  chloride o .  i  gramme 

Ammonium  tartrate       10.0  grammes 

and  dissolve  in 

Distilled  water 1000  c.c. 

2.  Tube  or  flask;  sterilise  as  for  nutrient  bouillon. 


192  SPECIAL   MEDIA 

Plaster=of -Paris  Discs.— 

i.  Take  large  corks,  2.5  cm.  diameter,  and  roll  a  piece  of  stiff 
note-paper  round  each,  so  that  about  a  centimetre  projects  as  a 
ridge  above  the  upper  surface  of  the  cork,  and  secure  in  position 
with  a  pin  (Fig.  112). 

2.  Mix    plaster-of -Paris   into    a    stiff    paste 
with  distilled  water,  and  fill  each  of  the  cork 
moulds  with  the  paste. 

3.  When  the  plaster  has  set,   remove  the 
paper  from  the  corks,   and  raise  the  plaster 
discs. 

4.  Place    the    plaster    discs    on    a    piece  of 
asbestos  board  and  sterilise  by  exposing  in  the 
hot-air  oven  to  150°  C.  for  half  an  hour. 

Fig.  112. — Cork  5.  Remove  the  sterile  discs  from  the  oven 
and  paper  mould  for  by  means  of  sterile  forceps,  place  each  inside 
plaster-of-Paris  disc,  a  sterile  capsule,  and  moisten  with  a  little 
sterile  water. 

6.  Sterilise  in  the  steamer  at  100°  C.  for  thirty  minutes  on 
each  of  three  consecutive  days. 

Gypsum  Blocks  (Engel  and  Hansen). — 

These  are  in  the  form  of  truncated  cones  and  for  their  prepara- 
tion small  tin  moulds  are  required,  each  having  a  diameter  of  5 . 5 
cm.  at  the  base  and  4  cm.  at  the  truncated  apex.  The  height 
(or  depth)  of  a  mould  is  4 . 5  to  5  cm. 

1.  Mix  powdered  calcined  gypsum  into  a  stiff  paste  with  dis- 
tilled water. 

2.  Fill  the  paste  into  the  moulds  and  allow  it  to  set  and  dry  by 
exposure  to  air. 

3.  Remove    the    block  from  the  mould  and  transfer  it  to    a 
double  glass  dish  of  adequate  size  (7  cm.  diameter X  7  cm.  high). 

4.  Sterilise  block  in  its  dish  for  one  hour  in  the  hot-air  oven  at 
115°  C. 

5.  Carefully  open  the  dish  and  add  sterile  distilled  water  to 
moisten  the  block  and  form  a  layer  in  the  bottom  of  the  dish  i  cm. 
deep. 

Wine  Must. — (Wine  must  is  obtained  from  Sicily,  in  hermetically 
sealed  tins,  in  a  highly  concentrated  form — as  a  thick  syrup — • 
but  not  sterilised.) 

1.  Weigh  out  "wine  must,"   200  grammes,  place  in  a   2-litre 
flask  and  add.  distilled  water,  800  c.c. 

2.  Weigh  out  ammonium  tartrate,  5  grammes,  and  add  to  the 
dilute  must. 

3.  Place  the  flask  in  a  water-bath  regulated  to  60°  C.  for  one 
hour  and  incorporate  the  mixture  thoroughly  by  frequent  shaking. 

4.  Filter  through  papier  Chardin. 

5.  Tube,  and  sterilise  as  for  nutrient  bouillon. 


GELATINE    AGAR  193 

Wheat  Bouillon  (Gasperini).— 

1.  Weigh  out  and  mix  wheat  flour,  150  grammes;  magnesium 
sulphate,    0.5   gramme;   potassium  nitrate,    i    gramme;   glucose, 
15  grammes. 

2.  Dissolve  the  mixture  in  1000  c.c.  of  water  heated  to  100°  C. 

3.  Filter  through  papier  Chardin. 

4.  Tube,  and  sterilise  as  for  nutrient  bouillon. 

Bread  Paste. — 

1.  Grate  stale  bread  finely  on  a  bread-grater. 

2.  Distribute  the  crumbs  in  sterile  Erlenmeyer  flasks,  sufficient 
to  form  a  layer  about  one  centimetre  thick  over  the  bottom  of 
each. 

3.  Add  as  much  distilled  water  as  the  crumbs  will  soak  up, 
but  not  enough  to  cover  the  bread. 

4.  Plug  the  flasks  and  sterilise  in  the  steamer  at  100°  C.  for 
thirty  minutes  on  each  of  four  consecutive  days. 

Media  for  the  Study  of  Parasitic  Moulds. 

French  Proof  Agar  (Sabouraud). — 

1.  Weigh  out  Chassaing's  peptone,  10  grammes,  and  emulsify  it 
with  200  c.c.  distilled  water  previously  heated  to  60°  C. 

2.  Weigh  out  powdered  agar,  13  grammes,  and  emulsify  with 
200  c.c.  cold  distilled  water. 

3.  Mix  the  two  emulsions  and  wash  into  a  tared  2-litre  flask 
with  600  c.c.  distilled  water. 

4.  Bubble  live  steam  through  the  mixture  for  twenty  minutes, 
to  dissolve  the  agar. 

5.  Cool  to  60°  C.   and  clarify  with  egg  as  for  nutrient  agar 
(vide  page  168). 

6.  Filter  through  Papier  Chardin,  using  the  hot-water  funnel. 

7.  Weigh  out  French  maltose,  40  grammes,  and  dissolve  in  the 
agar. 

8.  Tube,  and  sterilise  as  for  nutrient  agar. 

English  Proof  Agar  (Blaxall).— Substitute  Witte's  peptone  for 
that  of  Chassaing,  and  proceed  as  for  French  proof  agar. 

French  Mannite  Agar,  Sabouraud. — (For  cultivation  of  Favus.) 
Proceed  exactly  as  in  preparing  French  Proof  agar  vide  supra 
substituting  Mannite  (38  grammes)  for  maltose. 

Media  for  the  Study  of  Milk  Bacteria. 

Gelatine  Agar. — This  medium  is  prepared  by  adding  to  nutrient 
gelatine  sufficient  agar  to  ensure  the  solidity  of  the  medium 
when  incubated  at  temperatures  above  22°  C.  If  it  is  intended 

13 


SPECIAL   MEDIA 

to  employ  an  incubating  temperature  of  30°  C.,  10  per  cent, 
gelatine  and  0.5  per  cent,  agar  must  be  dissolved  in  the  meat 
extract  before  the  addition  of  the  peptone  and  salt;  while  for 
incubating  at  37°  C.,  12  per  cent,  gelatine  and  0.75  per  cent,  agar 
must  be  used.  Avoid  the  addition  of  more  agar  than  is  absolutely 
necessary,  otherwise  the  action  upon  the  medium  of  such  organisms 
as  elaborate  a  liquefying  ferment  may  be  retarded  or  completely 
absent. 

1.  Measure  out  400   c.c.  double  strength  meat  extract  into  a 
"tared"  2-litre  flask,  and  add  to  it  gelatine,  100  grammes. 

2.  Weigh  out  powdered  agar,  5  grammes,  emulsify  with  100  c.c., 
cold  distilled  water  and  add  to  the  contents  of  the  flask. 

3.  Dissolve    the    agar    and   gelatine  by  bubbling  live   steam 
through  the  flask  for  twenty  minutes. 

4.  Weigh  out  peptone,  10  grammes;  salt,  5  grammes ;  emulsify 
with  100  c.c.  double  strength  meat  extract  previously  heated  to 
60°  C.,  and  add  to  the  contents  of  the  flask. 

5.  Replace  in  the  steamer  for  fifteen  minutes.     Then  adjust  the 
weight  to  the  calculated  figure  for  one  litre  (in  this  instance  1120 
grammes)  by  the  addition  of  distilled  water  at  100°  C. 

6.  Estimate    the    reaction;    control    the    result.     Then    add 
sufficient  caustic  soda  solution  to  render  the  reaction  +  10. 

7.  Replace  in  the  steamer  at  100°  C.  for  twenty  minutes. 

8.  Cool  to  60°  C.     Clarify  with  egg  as  for  nutrient  agar. 

9.  Filter  through  papier  Chardin,  using  the  hot-water  funnel. 

10.  Tube,  and  sterilise  as  for  nutrient  agar. 

Agar  Gelatine   (Guarniari). — 

1.  Measure  out  double  strength  meat  extract,  400  c.c.,  into  a 
"tared"  2-litre  flask,  and  add  to  it  gelatine,  50  grammes. 

2.  Weigh  out  powdered  agar,  3  grammes;  emulsify  with  cold 
distilled  water,  50  c.c.,  and  add  to  the  contents  of  the  flask. 

3 .  Dissolve  the  agar  and  gelatine  by  bubbling  live  steam  through 
the  flask  for  twenty  minutes. 

4.  Weigh  out  Witte's  peptone,  25  grammes;  salt,  5  grammes, 
and  emulsify  with  100  c.c.  double  strength  meat  extract  previously 
heated  to  60°  C.,  and  add  to  the  contents  of  the  flask. 

5.  Replace,  in  the  steamer  for  fifteen  minutes. 

6.  Weigh  the  flask  and  make  up  the  medium  mass  to  the  cal- 
culated figure  for  one  litre   (1083  grammes)   by  the  addition  of 
distilled  water  at  100°  C. 

7.  Neutralise    carefully    to    litmus    paper    by    the    successive 
additions  of  small  quantities  of  normal  soda  solution. 

8.  Replace  in  the  steamer  at  100°  C.  for  twenty  minutes. 

9.  Cool  to  60°  C.     Clarify  with  egg  as  for  nutrient  agar. 

10.  Filter  through  papier  Chardin,  using  the  hot-water  funnel. 

11.  Tube,  and  sterilise  as  for  nutrient  agar. 


LITMUS    WHEY 


195 


Whey  Gelatine. — 


1.  Curdle  fresh  milk  by  warming  to  60°  C.,  and  adding  rennet; 
filter  off  the  whey  into  a  sterile  "tared"  flask. 

2.  Estimate  and  note  the  reaction  of  the  whey. 

3.  Weigh  out  gelatine,   10  per  cent.,  and  add  it  to  the  whey 
in  the  flask. 

4.  Bubble  live  steam  through  the  mixture  fifteen  minutes  to 
dissolve  the  gelatine;  and  weigh. 

5.  Estimate    the    reaction    of    the    medium    mass;    then    add 
sufficient   caustic   soda   solution  to   restore   the   reaction   of  the 
medium  mass    (i.e.,  total  weight  minus  weight  of  flask)  to  the 
equivalent  of  the  original  whey. 

6.  Cool  to  60°  C.  and  clarify  with  egg  as  for  nutrient  gelatine 
(vide  page  166). 

7.  Filter  through  papier  Chardin. 

8.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

Whey  Agar. — 

1.  Curdle  fresh  milk  by  warming  to  60°  C.,  and  adding  rennet; 
filter  off  the  whey  into  a  sterile  flask. 

2.  Weieh  out  agar,  1.5  or  2  per  cent.,  and  add  it  to  the  whey 
in  the  flask. 

3.  Bubble  live  steam  through  the  mixture  for  twenty  minutes, 
to  dissolve  the  agar. 

4.  Cool  to  60°  C.;  clarify  with  egg  as  for  nutrient  agar  (vide 
page  1 68). 

5.  Filter  through  papier  Chardin,  using  the  hot-water  funnel. 

6.  Tube,  and  sterilise  as  for  nutrient  agar. 

Litmus  Whey.— 

1.  Curdle  fresh  milk  by  warming  to  60°  C.  and  adding  rennet. 

2.  Filter  off  the  whey  through  butter  muslin  into  a  sterile  flask. 

3.  Neutralise  to  litmus  by  the  cautious  addition  of  citric  acid 
solution  4  per  cent.      (Do  not  neutralise  with  mineral  acid.) 

4.  Heat  in  the  steamer  at  100°  C.  for  one  hour  to  coagulate  all 
the  proteid. 

(If  the  whey  is  cloudy  when  removed  from  the  steamer  allow 
it  to  stand  for  forty-eight  hours  in  the  ice  chest  and  then  decant 
off  the  clear  fluid — or  filter  through  a  Berkfeld  filter  candle.) 

5.  Filter  into  a  sterile  flask. 

6.  Tint  the  whey  with  litmus  solution  to  a  deep  purple  red. 

7.  Tube,  and  sterilise  as  for  milk. 

Litmus  Whey  (Petruschky).— 

i.   Measure  out  into  a  flask 

Fresh  milk  1000  c.c. 


196  SPECIAL   MEDIA 

2.  Add 

Hydrochloric  acid  (or  glacial  acetic  acid)  .    i .  5  c.c. 
and  boil. 

3.  Filter  off  coagulated  casein. 

4.  Neutralise  to  litmus  by  the  addition  of  5  caustic  soda   so- 
lution and  boil.     Whey  now  cloudy  and  acid  again. 

5.  Again  neutralise  to  litmus  by  addition  of  -^   caustic  soda 
solution. 

6.  Filter. 

7.  Tint  the  whey  with  neutral  litmus  solution  to  a  deep  purple 
colour. 

8.  Tube  and  sterilise  as  for  milk. 

Litmus  Whey  Gelatine.— 

1.  Measure  out  milk  1000  c.c.  into  a  tared  2-litre  flask. 

2.  Add  hydrochloric  acid   (or  glacial  acetic  acid)    1.5  c.c.  and 
boil  for  five  minutes. 

3.  Filter  off  the  casein,  and  make  the  whey  faintly  alkaline  to 
litmus. 

4.  Weight  out 

Peptone 10  grammes 

and  emulsify  in  a  few  cubic  centimeters  of  the  whey  and  return 
to  the  flask. 

5.  Weight  out 

Gelatine 50  grammes 

add  it  to  the  whey  in  the  flask  and  incorporate  the  mixture 
by  bubbling  through  live  steam. 

6.  Clear  with  egg  and  filter. 

7.  Make  the  weight  of  the  medium  mass  to  the  calculated  fig- 
ure for  one  litre  (1060  grammes)  by  the  addition  of  distilled  water. 

8.  Weigh  out 

Dextrose       15  grammes 

and  dissolve  in  the  fluid  whey  gelatine. 

9.  Add  sterile  litmus  solution  to  the  required  tint. 

10.  Tube  and  sterilise  for  twenty  minutes  in  steamer  at  100°  C. 
on  each  of  five  successive  days. 

This  medium  will  remain  semi-fluid  at  the  room  temperature, 
and  may  be  used  for  cultures  in  the  cool  or  hot  incubator. 

Litmus  Whey  Agar  is  prepared  in  a  similar  manner  to  Whey 
Gelatine,  with  the  substitution  of  15  grammes  of  agar  for  the 
gelatine. 

Malt  Extract  Solution  (Herschell)  .— 

1.  Measure  into  a  flask  distilled  water  1000  c.c. 

2.  Weigh  out 

Extractum  malti  (malt  extract)  .    .    25  grammes 
and  add  to  distilled  water  in  flask. 


BEYRINCK'S  SOLUTION  197 

3.  Boil  for  five   minutes,   allow  to  stand,  and  decant  off  clear 
fluid  from  sediment. 

4.  Tube  and  sterilise  as  for  nutrient  bouillon. 

Media  for  the  Study  of  Earth  Bacteria,  Nitrogen  Fixers. 

Earthy  Salts  Agar  (Lipman  and  Brown). — (For  the  enumeration 
of  soil  organisms.} 

1.  Measure  out 

Agar     .'.... 20  grammes. 

Emulsify  in  200  c.c.  distilled  water. 

2.  Wash  the  agar  emulsion  into  a  tared  2-litre  flask  with  400  c.c. 
distilled  water. 

3.  Weigh  out 

Peptone 0.5  gramme. 

Emulsify  in  50  c.c.  distilled  water  and  add  to  the  contents 
of  the  flask. 

4.  Bubble  live  steam  through  the  mixture  for  twenty  minutes 
to  dissolve  the  agar. 

5.  Weigh  out  and  mix 

Dextrose 10.0    grammes. 

Potassium  phosphate     ....  0.5    gramme. 

Magnesium  sulphate 0.2     gramme. 

Potassium  nitrate 0.06  gramme. 

and  add  to  the  contents  of  the  flask. 

6.  Adjust  the  weight  of  the  medium  mass  to  the  calculated  fig- 
ure for  one  litre  (1025  grammes)  by  the  addition  of  distilled  water 
at  100°  C. 

7.  Titrate  the  medium  mass  and  adjust  the  reaction  to  +5. 

8.  Cool  to  60°  C.     Clarify  with  egg  and  filter. 

9.  Tube  in  quantities  of  10  c.c.  and  sterilise  as  for  nutrient  agar. 

Beyrinck's  Solution.  I. — (For    the  cultivation  of  nitrogen  fixing 
organisms.) 

1.  Weigh  out  and  mix   i   gramme  potassium  hydrogen  phos- 
phate, 0.2  gramme  magnesium  sulphate,  and  0.02  gramme  sodium 
chloride. 

2.  Dissolve  in  water  1000  c.c.,  in  a  2 -litre  flask. 

3.  Add  i  c.c.  of  a  one  per  thousand  aqueous  solution  of  ferrous 
sulphate. 

4.  Add     i    c.c.    of    a   one   per   thousand  solution  manganese 
sulphate. 

5.  Weigh  out  20  grammes  dextrose  and  add  to  the  contents  of 
the  flask  (dextrose  up  to  40  grammes  may  be  used  for  the  different 
organisms) . 

6.  Steam  for  twenty  minutes,  filter. 

7.  Tube,  and  sterilise  as  for  nutrient  bouillon. 


198  SPECIAL   MEDIA 

Beyrinck's  Solution.  II. — (For  growth  of  Azobacter.) 
Proceed  as  in  preparing  solution  No.  i,  substituting  mannite  for 
dextrose  in  step  5. 

Winogradsky's  Solution  (for  Nitric  Organisms) . — 

1.  Weigh  out  and  mix. 

Potassium  phosphate i .  o     gramme 

Magnesium  sulphate       0.5     gramme 

Calcium  chloride o.oi  gramme 

Sodium  chloride 2.0    grammes 

and  dissolve  in 

Distilled  water 1000  c.c. 

2.  Fill  into  flasks,  in  quantities  of  20  c.c.    and   add  to  each  a 
small  quantity  of  freshly  washed  magnesium  carbonate. 

3.  Sterilise  in  the  steamer  at  100°  C.  for  twenty  minutes  on 
each  of  three  consecutive  days. 

4.  Add  to  each  flask  containing  20  c.c.  solution,   2   c.c.  of  a 
sterile  2  per  cent,  solution  of  ammonium  sulphate. 

5.  Incubate  at  37°  C.  for  forty-eight  hours  and  eliminate  any 
contaminated  culture  flasks.     Store  the  remainder  for  future  use. 

Winogradsky's  Solution  (for  Nitrous  Organisms). — 

1.  Weigh  out  and  mix 

Ammonium  sulphate i  gramme 

Potassium  sulphate i  gramme 

and  dissolve  in 

Distilled  water 1000  c.c. 

2.  Add  5  to  10  grammes  basic  magnesium  carbonate,  previously 
sterilised  by  boiling. 

3.  Fill  into  flasks  and  sterilise,  etc.,  as  for  previous  solution. 

Silicate  Jelly  (Winogradsky).— 

1.  Weigh  out  and  mix 

Ammonium  sulphate 0.40  gramme 

Magnesium  sulphate       0.05  gramme 

Calcium  chloride o.oi  gramme 

and  dissolve  in 

Distilled  water 50  c.c. 

Label — Solution  A. 

2.  Weigh  out  and  mix 

Potassium  phosphate o.io  gramme 

Sodium  carbonate       0.60  gramme 

and  dissolve  in 

Distilled  water 50  c.c. 

Label — Solution  B. 


BILE    SALT    BROTH  199 

3.  Weigh  out 

Silicic  acid 3.4  grammes 

and  dissolve  in 

Distilled  water 100  c.c. 

4.  Pour  the  silicic  acid  solution  into  a  large  porcelain  basin. 

5.  Mix  equal  quantities  of  the  solutions  A  and   B;  then  add 
successive  small  quantities  of  the  mixed  salts  to  the  silicic  acid 
solution,  stirring  continuously  with  a  glass  rod,  until  a  jelly  of 
sufficiently  firm  consistence  has  been  formed. 

6.  Spread  a  layer  of  this  jelly  over  the  bottom  of  each  of  several 
large  capsules  or  "plates." 

7.  Sterilise  in  the  steamer  at  100°  C.  for  thirty  minutes  on  each 
of  three  consecutive  days. 

Media  for  the  Study  of  Water  Bacteria. 

Naehrstoff  Agar  (Hesse  and  Niedner) . — (For  enumeration  of  water 
organisms.) 

1.  Weigh  out:  agar,    12.5   grammes  and  emulsify  in   250  c.c. 
distilled  water. 

2.  Wash  the  agar  emulsion  into  a  tared   2 -litre  flask  with  a 
further  250  c.c.  distilled  water. 

3.  Dissolve  by  bubbling  live  steam  through  the  mixture. 

4.  Emulsify    Naehrstoff-Heyden    (albumose)    7 . 5   grammes   in 
200  c.c.  cold  distilled  water  and  add  to  melted  agar. 

5.  Adjust  weight  of  medium  mass  to  the  calculated  figure  for  one 
litre  (1020  grammes)  by  addition  of  distilled  water  at  100°  C. 

6.  Clarify  with  white  of  egg  and  filter. 

7.  Tube  in  quantities  of  10  c.c.  and  sterilise  in  the  steamer  at 
1 00°  C.  for  twenty  minutes  on  each  of  three  successive  days. 

Bile  Salt  Broth— Double  Strength.— 

1.  Weigh  out  Witte's  peptone,  40  grammes,  and  emulsify  with 
300  c.c.  distilled  water  previously  warmed  to  60°  C. 

2.  Wash  the  peptone  emulsion 'into  a  litre  flask  with  600  c.c. 
distilled  water. 

3.  Weigh  out  sodium  taurocholate,    10  grammes,  and  glucose, 
10  grammes;  dissolve  in  100  c.c.  distilled  water  and  add  to  the 
peptone  emulsion  in  the  flask. 

4.  Heat  in  the  steamer  at  100°  C.  for  twenty  minutes. 

5.  Filter  through  Swedish  filter  paper  into  a  sterile  flask. 

6.  Add   sterile  neutral  litmus  solution  sufficient  to  colour  the 
medium  to  a  deep  purple. 

7.  Fill  into  small  Erlenmeyer  flasks  in  quantities  of  25  c.c. 

8.  Sterilise  as  for  nutrient  bouillon. 


200  SPECIAL   MEDIA 

Media  for  the  Study  of  Plant  Bacteria. 

Beetroot.—  1 

Carrot. —      [  are  prepared  tubes  and  sterilised  in  a  manner  pre- 
Turnip. —      (  cisely  similar  to  that  described  for  potato. 
Parsnip. —   J 

Hay  Infusion. — 

1.  Weigh   out   dried   hay,    10   grammes,    chop   it   up   into  fine 
particles  and  place  in  a  flask. 

2.  Add   1000  c.c.  distilled  water,  heated  to  70°  C.;  close  the 
flask  with  a  solid  rubber  stopper. 

3.  Macerate  in  a  water-bath  at  60°  C.  for  three  hours. 

4.  Replace  the  stopper  by  a  cotton-wool   plug,   and    heat  in 
the  steamer  at  100°  C.  for  one  hour. 

5.  Filter  through  Swedish  filter  paper. 

6.  Tube,  and  sterilise  as  for  nutrient  bouillon. 

Haricot  Bouillon. — (For  cultivation  of  bacteria  from  tubercles  of 
Legumes.) 

1.  Measure  out  1000  c.c.  distilled  water  into  a  2 -litre  flask. 

2.  Weigh  out  250  grammes  haricot  beans  and  add  to  the  water 
in  the  flask. 

3.  Weigh   out    10   grammes   sodium   chloride   and   add   to  the 
contents  of  the  flask. 

4.  Add  i  c.c.  of  a  i  per  cent,  solution  of  sodium  bicarbonate. 

5.  Place  in  the  steamer  at  100°  C.  for  thirty  minutes. 

6.  Filter. 

7.  Weigh  out  20  grammes  saccharose  and  add  to  the  filtrate. 

8.  Tube,  and  sterilise  as  for  nutrient  bouillon. 

Haricot  Agar. — 

1.  Measure   out  400  c.c.  distilled  water  into  a  "tared"    2-litre 
flask. 

2.  Weigh  out  15  grammes  agar  and  mix  into  a  thick  paste  with 
100  c.c.  cold  distilled  water,  and  add  to  the  flask. 

3.  Dissolve  the  agar  by  bubbling  live  steam  through  the  mixture 
as  in  making  nutrient  agar. 

4.  Weigh  out  250  grammes  haricot  beans,  place  in  the  flask  with 
the  agar  mixture. 

5.  Add  i  c.c.  of  i  per  cent,  aqueous  solution  sodium  bicarbonate. 

6.  Weigh   out    10   grammes   sodium   chloride   and   add    to    the 
contents  of  the  flask. 

7.  Place  in  the  steamer  at  100°  C.  for  thirty  minutes. 

8.  Adjust  the  weight  of  the  medium  mass  to  1030  grammes  (the 
figure  per  litre  obtained  experimentally)  by  the  addition  of  distilled 
water  at  100°  C. 


OLE  1C   ACID   AGAR  2OI 

9.  Cool  to  60°  C.,  clarify  with  egg  and  filter. 

10.  Weigh  out  20  grammes  saccharose  and  add  to  the  contents 
of  the  flask. 

11.  Tube,  and  sterilise  as  for  nutrient  agar. 

Wood  Ash  Agar.— 

1.  Measure  400  c.c.  distilled  water  into  a  tared  2-litre  flask. 

2.  Weigh  out  10  grammes  agar  and  make  into  a  thick  paste 
with  100  c.c.  cold  distilled  water. 

3.  Add  this  agar  paste  to  the  distilled  water  in  the  flask. 

4.  Dissolve  the  agar  by  passing  live  steam  through  it,  as  in 
preparing  nutrient  agar. 

5.  Weigh  out  5  grammes  clean  wood  ash  and  place  in  a  second 
flask  containing  200  c.c.  distilled  water  with  some  sterile  glass 
beads:  shake  thoroughly  in  a  mechanical  shaker  for  ten  minutes. 

6.  Heat  in  steamer  at  100°  C.,  for  thirty  minutes. 

7.  After  removal  from  the  steamer  dry  the  outside  of  the  flask 
thoroughly,  place  it  over  a  Bunsen  flame  and  boil  for  one  minute. 

8.  Filter  directly  into  the   flask    containing   the   melted   agar 
mixture. 

9.  Weigh  out  4  grammes  maltose.     Add  to  the  contents  of  the 
flask. 

10.  Adjust  the  weight  of  the  medium  mass  to  the    calculated 
figure  for  one  litre  (1019  grammes)  by  the  addition  of   distilled 
water  at  100°  C. 

11.  Replace  the  flask  in  the  steamer  for  twenty  minutes,  cool 
to  60°  C.,  and  clarify  with  egg  and  filter. 

12.  Tube,  and  sterilise  as  for  nutrient  agar. 

Media  for  the  Study  of  Special  Bacilli. 

B.  Acnes. 
Oleic  Acid  Agar  (Fleming).— 

1.  Measure  out  into  a  sterile  stout  glass  bottle  which  already 
contains  about  10  sterile  glass  beads 

Ascitic  fluid 250  c.c. 

2.  Weigh  out 

Oleic  acid 25  grammes 

and  add  it  to  the  ascitic  fluid  in  the  bottle. 

3.  Emulsify  evenly  by  shaking  (either  by  hand  or  in  a  shaking 
machine)  for  ten  minutes. 

4.  Liquefy  and  measure  out  into  a  flask 

Nutrient  agar 750  c.c. 

then  cool  to  55°  C. 

5.  Mix  the  oleic  acid  emulsion  with  the  agar. 


202  SPECIAL   MEDIA 

6.  Add  10  c.c.  sterile  neutral  red,  i  per  cent,  aqueous  solution. 

7.  Tube  in  quantities  of  10  c.c.,  slant,  and  allow  to  set. 

8.  Incubate   for  forty-eight  hours   at   37°    C.    and   reject   any 
contaminated  tubes.     Store  the  sterile  tubes  for  future  use. 

Coli-typhoid  Group. 
Parietti's  Bouillon.— 

1.  Measure  out  pure  hydrochloric  acid,  4  c.c.,  and  add  to  it 
carbolic  acid  solution  (5  per  cent.),  100  c.c.     Allow  the  solution 
to  stand  at  least  a  few  days  before  use. 

2.  This  solution  is  added  in  quantities  of  o.i,  0.2.  and  0.3  c.c. 
(delivered  by  means  of  a  sterile  graduated  pipette)  to  tubes  each 
containing  10  c.c.  of  previously  sterilised  nutrient  bouillon  (vide 
page  163). 

3.  Incubate  at  37°  C.  for  forty-eight  hours  to  eliminate  con- 
taminated tubes.     Store  the  remainder  for  future  use. 

Carbolised  Bouillon. — 

1.  Prepare  nutrient  bouillon   (vide  page  163,  sections    i   to  6). 
Measure  out  1000  c.c. 

2.  Weigh  out  carbolic  acid,  i  gramme  (2  .  5  or  5  grammes  may 
be  needed  for  special  purposes),  and  dissolve  it  in  the  medium. 

3.  Tube,  and  sterilise  as  for  bouillon. 

Carbolised  Gelatine.— 

1.  Prepare  nutrient  gelatine   (vide  page  164,  sections   i   to  7). 
Measure  out  1000  c.c. 

2.  Weigh  out  carbolic  acid,  5  grammes  (  =  0.5  per  cent.),  and 
dissolve  it  in  the  gelatine. 

3.  Filter  if  necessary  through  papier  Chardin. 

4.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

One  or  2.5  grammes  of  carbolic  acid  (  =  0.1  per  cent,  or  0.25 
per  cent.)  are  occasionally  used  in  place  of  the  5  grammes  to  meet 
special  requirements.  • 

Carbolised  Agar. — 

1.  Prepare   nutrient   agar    (vide    page    167,    sections    i    to    8). 
Measure  out  1000  c.c. 

2.  Weigh  out  i  gramme  pure  phenol  and  dissolve  in  the  medium. 

3 .  Filter  if  necessary  through  papier  chardin. 

4.  Tube,  and  sterilise  as  for  nutrient  agar. 

Litmus  Gelatine. — 

i.  Prepare  nutrient  gelatine  (vide  page  164,  sections  i  to  8). 
2-.  Add  sterile  litmus  solution,  sufficient  to  tint  the  medium  a 
deep  lavender  colour. 

3.  Tube,  and  sterilise  as  for  nutrient  gelatine. 


GLYCERINE    POTATO    BOUILLON  203 

Lactose  Litmus  Bouillon   (Lakmus  Molke). — 

1.  Weight    out    peptone,    4    grammes,    and   emulsify  it   with 
200  c.c.  meat  extract  (vide  page  148),  previously  heated  to  60°  C. 

2.  Weigh  out  salt,  2  grammes,  and  lactose,  20  grammes,  and  mix 
with  the  emulsion. 

3.  Wash  the  mixture  into  a  sterile  litre  flask  with   200   c.c. 
meat  extract  and  add  600  c.c.  distilled  water. 

4.  Heat  in  the  steamer  at  100°  C.  for  thirty  minutes,  to  com- 
pletely dissolve  the  peptone,  etc. 

5.  Neutralise  carefully  to  litmus  paper  by  the  successive  additions 
of  small  quantities  of  decinormal  soda  solution. 

6.  Replace  in  the  steamer  for  twenty  minutes  to  precipitate 
phosphates,  etc. 

7.  Filter  through  two  thicknesses  of  Swedish  filter  paper. 

8.  Add  sterile  litmus  solution,  sufficient  to  colour  the  medium 
a  deep  purple. 

9.  Tube,  and  sterilise  as  for  bouillon. 

Lactose  Litmus  Gelatine  (Wurtz).— 

1.  Prepare  nutrient  gelatine  (vide  page  164,  sections  i  to  4). 

2.  Render  the  reaction  of  the  medium  mass  — 5. 

3.  Replace  in  the  steamer  at  100°  C.  for  twenty  minutes. 

4.  Clarify  with  egg  as  for  gelatine. 

5.  Weigh  out  lactose,  20  grammes  (  =  2  per  cent.),  and  dissolve 
it  in  the  medium. 

6.  Filter  through  papier  Chardin. 

7.  Add  sufficient  sterile  litmus  solution  to  colour  the  medium 
pale  lavender. 

8.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

Lactose  Litmus  Agar  (Wurtz). — 

1.  Prepare  nutrient  agar  (vide  page  167,  sections  i  to  4). 

2 .  Render  the  reaction  of  the  medium  mass  —  5 . 

3.  Replace  in  the  steamer  at  100°  C.  for  twenty  minutes. 

4.  Cool  to  60°  C.  and  clarify  with  egg  as  for  nutrient  agar. 

5.  Weigh  out  lactose,  20  grammes  (  =  2  per  cent.),  and  dissolve 
it  in  the  medium. 

6.  Filter  through  papier  Chardin,  using  the  hot-water  funnel. 

7.  Add  sterile  litmus  solution,  sufficient  to  colour  the  medium 
a  pale  lavender. 

8.  Tube,  and  sterilise  as  for  nutrient  agar. 

Glycerine  Potato  Bouillon.— 

1.  Take    i    kilo  of  potatoes,   wash  thoroughly  in  water,   peel, 
and  grate  finely  on  a  bread-grater. 

2.  Weigh  the  potato   gratings,    place    them  in  a  2 -litre  flask, 


204  SPECIAL   MEDIA 

and  add  distilled  water  in  the  proportion  of  i  c.c.  for  every 
gramme  weight  of  potato.  Allow  the  flask  to  stand  in  the  ice- 
chest  for  twelve  hours. 

3.  Strain  the  mixture  through  butter  muslin  and  filter  through 
Swedish  filter  paper  into  a  graduated  cylinder.     Note  the  amount 
of  the  filtrate. 

4.  Place  the  filtrate  in  a  flask,  add  an  equal  quantity  of  dis- 
tilled water,  and  heat  in  the  steam  steriliser  for  sixty  minutes. 

5.  Add    glycerine,    4    per    cent.,    mix    thoroughly,    and    again 
filter. 

6.  Tube  and  sterilise  as  for  nutrient  bouillon. 

Potato  Gelatine  (Eisner).— 

1.  Take    i   kilo  of  potatoes,   wash  thoroughly  in  water,  peel, 
and  finally  grate  finely  on  a  bread-grater. 

2.  Weigh  the   potato   gratings,    place  them   in    a   2 -litre   flask, 
and  add  distilled  water  in  the  proportion  of  i  c.c.  for  every  gramme 
weight  of  potato.     Allow  the  flask  to  stand  in  the  ice-chest  for 
twelve  hours. 

3.  Strain  the  mixture  through  butter  muslin,  and  filter  through 
Swedish  filter  paper  into  a  graduated  cylinder. 

4.  Add    15    per   cent,    gelatine   to   the   potato   decoction    and 
bubble  live  steam  through  the  mixture  for  ten  minutes. 

5.  Estimate  the  reaction;  adjust  the  reaction  of  the  medium 
mass  to  +25. 

6.  Cool  the  medium  to  below  60°  C.;  clarify  with  egg  as  for 
nutrient  gelatine  (vide  page  166). 

7.  Add  i  percent,  potassium  iodide  (powdered)  to  the  medium. 

8.  Filter  through  papier  Chardin. 

9.  Tube  and  sterilise  as  for  nutrient  gelatine. 

Aesculin    Agar. — (B.    coli    and     allied    organisms    give    black 
colonies  surrounded  by  black  halo.) 

1.  Measure  out  400  c.c.  distilled  water  into  a  tared  2-litre  flask. 

2.  Weigh  out 

Agar 15  grammes 

Peptone 10  grammes 

Sodium  taurocholate 5  grammes 

and  make  into  a  thick  paste  with  150  c.c.  distilled  water. 

3.  Add  this  paste  to  the  distilled  water  in  the  flask. 

4.  Dissolve  the  ingredients  by  bubbling  live  steam  through  the 
mixture. 

5.  Weigh  out 

Aesculin i .  o  gramme 

Ferric  citrate 0.5  gramme 

and  dissolve  in  a  second  flask  containing  100  c.c.  distilled  water. 

6.  Mix  the  contents  of  .the  two  flasks — adjust  the  weight  to 


FUCHSIN   A  GAR  205 

the  calculated  medium  figure  (in  this  case  1031.5  grammes)  by 
the  addition  of  distilled  water  at  100°  C. 

7.  Clarify  with  egg  and  filter. 

8.  Tube  and  sterilise  as  for  nutrient  agar. 

Bile  Salt  Agar  (MacConkey).— 

1.  Weigh  out  powdered  agar,    15   grammes  (  =  1.5.  per  cent.)' 
and  emulsify  with  200  c.c.  cold  tap  water. 

2.  Weigh  out  peptone,  20  grammes  ( =  2  per  cent.) ,  and  emulsify 
with  200  c.c.  tap  water  previously  warmed  to  60°  C. 

3.  Mix  the  peptone  and  agar  emulsions  thoroughly. 

4.  Weigh    out    sodium    taurocholate,    5    grammes    (  =  0.5    per 
cent.),    dissolve    it  in  300  c.c.   tap  water,  and  use  the    solution 
to  wash  the  agar-peptone  emulsion  into  a  tared  2-litre  flask. 

5.  Bubble  live  steam  through  the  mixture  for  twenty  minutes. 

6.  Adjust  the  weight  of  the  medium  mass   to   the    calculated 
figure  for  one  litre  (1040  grammes). 

7.  Cool  to  60°  C.   and  clarify  with  egg    as  for   nutrient   agar 
(vide  page  168). 

8.  Filter  through  papier  Chardin,  using  the  hot-water  funnel. 

9.  Weigh  out  lactose,  10  grammes  (=  i  per  cent.),  and  dissolve 
it  in  the  agar. 

If  desired,  add  5  c.c.  of  a  i  per  cent.  (  =  0.5  per  cent.)  aqueous 
solution  of  neutral  red. 
10.  Tube,  and  sterilise  as  for  nutrient  agar. 

Litmus  Nutrose  Agar  (Drigalski=Conradi)  .— 

This  medium  should  be  prepared  in  precisely  the  same  manner 
as  the  Nutrose  agar  described  on  page  172  substituting  meat  ex- 
tract for  serum  water,  and  increasing  the  percentage  of  agar  added 
per  litre  to  3  per  cent. 

Fuchsin  Agar  (Braun). — 

1.  Liquefy  and  measure  out  into  a  sterile  flask: 

Nutrient  agar 1000  c.c. 

2.  Weigh   out:   lactose    10   grammes  and  dissolve  in  the  fluid 
agar. 

3.  Adjust  the  reaction  to  -  5  and  filter. 

4.  Measure  out  and  mix  thoroughly  with  agar: 

Fuchsin,  alcoholic  solution 5  c.c. 

The  fuchsin  solution  is  prepared  by  mixing: 

Fuchsin  (basic) 3  grammes. 

Absolute  alcohol 60  c.c. 

Allow  to  stand  twenty-four  hours,  then  centrifugalise 
thoroughly  and  decant  the  supernatant  fluid  into  a 
well-stoppered  bottle,  j 


206  SPECIAL   MEDIA 

5.  Measure  out  and  add  to  the  nutrient  agar,  sodium  sulphite, 
10  per  cent,  aqueous  solution,  freshly  prepared   .    .    25  c.c. 

6.  Tube  and  sterilise  as  for  nutrient  agar. 

7.  Store  in  a  dark  cupboard. 

Fuchsin  Sulphite  Agar  (Endo).— 

1.  Liquefy  and  measure  out  into  a  sterile  flask: 

Nutrient  agar 1000  c.c. 

2.  Weigh  out 

Lactose 10  grammes. 

and  dissolve  in  the  fluid  agar. 

3.  Adjust  the  reaction  to  +3  and  filter. 

4.  Measure  out  and  mix  thoroughly  with  the  fluid  agar. 

Fuchsin,  alcoholic  solution  (vide  supra)     ....    5  c.c. 

5.  Measure  out  and  add  to  the  medium 

Sodium  sulphite,  10  per  cent,  aqueous  solution    .    .    25  c.c. 

6.  Tube  and  sterilise  as  for  nutrient  agar. 

Brilliant  Green  Agar  (Conradi)  .— 

1.  Liquefy  and  measure  out  into  a  sterile  flask 

Nutrient  agar 1000  c.c. 

2.  Adjust  reaction  to  +  30  by  the  addition  of  normal  phosphoric 
acid;  and  filter. 

3.  Measure  out  and  mix  thoroughly  with  the  fluid  medium 

Brilliant  green  (Hoechst)  i  per  thousand  aque- 
ous solution 6.5  c.c. 

4.  Measure  out  and  add  to  the  medium 

Picric  acid  (Gruebler),  i  per  cent,  aqueous  solution.    6.  5  c.c. 

5.  Tube  and  sterilise  as  for  nutrient  agar. 

Brilliant  Green  Bile  Salt  Agar  (Fawcus).— 

1.  Weigh  out  agar  20  grammes  and  emulsify  in  100  c.c.  cold 
distilled  water. 

2.  Wash  the  emulsion  into  a  "tared"  2-litre  flask  with  500  c.c. 
distilled  water. 

3.  Dissolve  the  agar  by  bubbling  live  steam  through  the  flask. 

4.  Cool,  clarify  with  egg  and  filter. 

5.  Weigh  out 

Sodium  taurocholate 5  grammes 

Peptone 20  grammes 

and  add  to  the  medium  in  the  flask. 


DOUBLE    SUGAR   AGAR  207 

6.  Weigh  out 

Lactose 5  grammes 

and  add  to  the  medium  in  the  flask. 

7.  Adjust  reaction  to  +   15  and  filter  if  necessary. 

8.  Measure  out 

Brilliant  green,  i  per  thousand  aqueous  solution .    20  c.c. 
and  mix  thoroughly  with  the  fluid  agar. 

9.  Measure  out  and  add  to  the  medium 

Picric  acid,  i  per  cent,  aqueous  solution    ....    20  c.c. 

10.  Tube  and  sterilise  as  for  nutrient  agar. 
China  Green  Agar  (Werbitski) . — 

1.  Liquefy  and  measure  out  into  a  sterile  flask 

Nutrient  agar 1000  c.c. 

2.  Adjust  the  reaction  accurately  to  +  13  and  filter. 

3.  Measure  out  and  mix  thoroughly  with  the  fluid  agar 
China  green  0.2  per  cent,  aqueous  solution.    .    .    15  c.c. 

4.  Tube  and  sterilise  as  for  nutrient  agar. 

Malachite  Green  Agar  (Loeffler). — 

1.  Liquefy  and  measure  out  into  a  sterile  flask 

Nutrient  agar 1000  c.c. 

2.  Weigh  out 

Dextrose      10  grammes. 

and  dissolve  in  nutrient  agar. 

3.  Adjust  the  reaction  to  +3,  and  filter. 

4.  Measure  out  and  mix  thoroughly  in  the  fluid  agar 

Malachite  green,  o.i  per  cent,  aqueous  solution.  .16  c.c. 
for  "weak"  medium. 

40.  To  the  filtered  agar  add 

Malachite  green,  2  per  cent,  aqueous  solution  . .  .25  c.c. 
for  "  strong  "  medium. 

5.  Tube  and  sterilise  as  for  nutrient  agar. 
Double  Sugar  Agar  (Russell). — 

1.  Liquefy  and  measure  out  into  a  sterile  flask 

Nutrient  agar 1000  c.c. 

2.  Add  100  c.c.  litmus  solution  to  the  fluid  agar. 

3.  Weigh  out  and  dissolve  in  the  fluid  agar. 

Lactose 10  grammes 

Dextrose  10  grammes. 


2O8  SPECIAL   MEDIA 

4.  Render  the   reaction    of    the     medium     neutral    to    litmus 
paper  by  the  cautious  addition  of  normal  caustic  soda. 

5.  Tube  in  quantities  of  10  c.c.  and  sterilise  in  the  steamer  at 
1 00°  C.  for  twenty  minutes  on  each  of  three  successive  days. 

6.  Store  for  use  in  a  cool  dark  place. 

B.  Diphtheria. 

Glycerine  Blood«=serum. — 

1.  Prepare  blood-serum  as  described,  page  168,  sections  i  to  4. 

2.  Add  5  per  cent,  pure  glycerine. 

3.  Complete    as    described    above    for    ordinary    blood-serum, 
sections  5  to  7. 

NOTE. — Different  percentages  of  glycerine — from  4  per  cent, 
to  8  per  cent.— are  used  for  special  purposes.  Five  per  cent,  is 
that  usually  employed. 

Blood  =serum   (Loeffler). — 

1.  Prepare    nutrient    bouillon    (vide    page     163),    using    meat 
extract  made  from  veal  instead  of  beef. 

2.  Add    i   per  cent,  glucose  to  the  bouillon,   and   allow  it  to 
dissolve  completely. 

3.  Now  add  300  c.c.  clear  blood-serum  (vide  page  168,  sections 
i  to  4)  to  every  100  c.c.  of  this  bouillon. 

4.  Fill  into  sterile  tubes  and  complete  as  for  ordinary  blood- 
serum. 

Blood =serum  (Lorrain  Smith). — 

1.  Collect  blood-serum  (vide  page  168,  sections  i  to  4),  as  free 
from  haemoglobin  as  possible. 

2.  Weigh  out  0.15  per  cent,  sodium  hydrate  and  dissolve  it 
in  the  fluid   (or  add  0.375  c.c.  of  dekanormal  soda  solution  for 
every  100  c.c.  of  serum). 

3.  Tube,  and  stiffen  at  100°  C.  in  the  serum  inspissator. 

4.  Incubate  at  37°  C.  for  forty-eight  hours  to  eliminate   any 
contaminated  tubes.     Store  the  remainder  for  future  use. 

Blood  Serum  (Councilman  and  Mallory). — 

1.  Collect  blood  serum  in   slaughterhouse,   coagulate,   remove 
serum  and  tube  (vide  page  168). 

Great  care  must  be  taken  to  avoid  the  inclusion  of  air  bubbles 
— indeed  if  only  a  few  tubes  are  filled  at  one  time,  it  is  a  good  plan 
to  stand  them  upright  in  the  receiver  of  an  air  pump  and  to  ex- 
haust as  completely  as  possible  before  transferring  to  the  serum 
inspissator. 

2.  Heat  the  tubes  in  a  slanting  position  in  hot-air  steriliser  at 
90°  C.  till  firmly  coagulated,  say  half  an  hour. 


GLYCERINATED    POTATO  209 

3.  Sterilise  in  steam  steriliser  at  100°  C.  for  20  minutes  on  each 
of  three  successive  days. 

Resulting  medium  not  translucent,  but  opaque  and  firm. 

B.  Tuberculosis. 

Egg  Medium  (Lubenau). — 

This  modification  of  Dorset's  egg  medium  (quod  inde  page 
174)  is  preferred  by  some  for  the  growth  of  the  tubercle  bacillus  of 
the  human  type.  It  consists  in  the  addition  of  one  part  of  6  per 
cent,  glycerine  in  normal  saline  solution,  to  the  egg  mixture  be- 
tween steps  4  and  5. 

Glycerine  Bouillon. — 

1.  Measure   out  nutrient  bouillon,    1000   c.c.    (vide  page    163, 
sections  i  to  6). 

2.  Measure  out  glycerine,  60  c.c.   (  =  6  per  cent.),  and  add  to 
the  bouillon. 

3.  Tube,  and  sterilise  as  for  bouillon. 

Glycerine  Agar. — 

1.  Prepare   nutrient    agar    (vide   page    167,    sections    i    to    8). 
Measure  out  1000  c.c. 

2.  Measure  out  pure  glycerine,  60  c.c.  (  =  6  per  cent.),  and  add 
to  the  agar. 

3.  Tube,  and  sterilise  as  for  nutrient  agar. 

Glycerine  Blood=serum.— 

1.  Prepare  blood-serum  as  described,  page  168,  sections  i  to  4. 

2 .  Add  5  per  cent,  pure  glycerine. 

3.  Complete  as  described  above  for  ordinary  blood-serum,  sec- 
tions 5  to  7. 

NOTE. — Different  percentages  of  glycerine — from  4  per  cent,  to 
8  per  cent. — are  used  for  special  purposes.  Five  per  cent,  is  that 
usually  employed. 

Glycerinated  Potato. — 

1.  Prepare    ordinary   potato   wedges    (vide  page    174,    sections 
i  to  4). 

2.  Soak  the  wedges  in  25  per  cent,  solution  of  glycerine  for 
fifteen  minutes. 

3.  Moisten  the  cotton- wool  pads  at  the  bottom  of  the  potato 
tubes  with  a  25  per  cent,  solution  of  glycerine. 

4.  Insert  a  wedge  of  potato  in  each  tube  and  replug  the  tubes. 

5.  Sterilise  in  the  steamer  at  100°  C.  for  twenty   minutes  on 
each  of  five  consecutive  days. 

14 


210  SPECIAL   MEDIA 

Animal  Tissue  Media  (Frugoni).— 

1.  Take  a  number  of  sterile  test-tubes  16X3  or  4  cm-»  plugged 
with  cotton  wool,  and  into  each  insert  a  2  cm.  length  of  stout  glass 
tubing    (about    i    cm.    diameter) ;   fill  in   glycerine    (6   per  cent.) 
bouillon  to  the  upper  level  of  the  piece  of  glass  tubing.     Sterilise 
in  the  steamer  at  100°  C.  for  twenty  minutes  on  each  of  three  suc- 
cessive days. 

2.  Kill  a  small  rabbit  by  means  of  chloroform  vapour. 

3.  Under  strictly  aseptic  precautions  remove  the  lungs,  liver  and 
other  solid  organs  and  transfer  them  to  a  sterile  double  glass  dish. 

4.  With  the  help  of  sterile  scissors  and  forceps  divide  the  organs 
into  roughly  rectangular  blocks  3X1.5X1  cm. 

5.  Pour  into  the  dish  a  sufficient  quantity  of  sterile  glycerine 
solution  (6  per  cent,  in  normal  saline),  cover,  and  allow  to  stand 
for  one  hour. 

6.  Introduce  a  block  of  tissue  into  each  tube  so  that  it  rests 
upon  the  upper  end  of  the  piece  of  glass  tubing.     (The  surface  of 
the  tissue  will  now  be  kept  moist  by  capillary  attraction  and 
condensation) . 

7.  Sterilise  in  the  autoclave  at  120°  C.  for  thirty  minutes. 

8.  Cap  the  tubes  and  store  them  in  the  ice  chest  for  future  use. 
Tissues  obtained  at  postmortems  can  also   be   used   after  pre- 
liminary sterilisation  by  boiling  or  autoclaving. 

Media  for  the  Study  of  Special  Cocci. 

Diplococcus  Gonorrhoea. 
Ascitic  Bouillon   (Serum  Bouillon). — 

1.  Collect  ascitic  fluid  (pleuritic  fluid,  hydrocele  fluid,  etc.),  by 
aspiration  directly  into  sterile  flasks,  under  strictly  aseptic  pre- 
cautions. 

2.  Mix  the  serum  with  twice  its  bulk  of  sterile  nutrient  bouillon 
(vide  page  163). 

3.  If    considered    necessary    (on    account    of    the    presence    of 
blood,  crystals,  etc.),  filter  the  serum  bouillon  through  porcelain 
filter  candle. 

4.  Tube,  and  sterilise  in  the  water  bath  at  56°  C.  for  half  an 
hour  on  each  of  five  consecutive  days. 

5.  Incubate  at  37°  C.  for  forty-eight  hours  and  eliminate  con- 
taminated tubes.     Store  the  remainder  for  future  use. 

Serum  Agar  (Heiman). — 

i.  Prepare  nutrient  agar  (vide  page  167),  to  following  formula: 

Agar 2.0  per  cent. 

Peptone 1.5  per  cent. 

Salt 0.5  per  cent. 

Meat  extract quantum  sufficit. 


SERUM   A GAR  211 

2.  Make  reaction  of  medium  +  10. 

3.  Filter;  tube  in  quantities  of  6  c.c. 

4.  Sterilise  as  for  nutrient  agar. 

5.  After  the  third  sterilisation  cool  the  tubes  to  42°  C.,  and 
add  to  each  3  c.c.  of  sterile  hydrocele  fluid,  ascitic  fluid,  or  pleuritic 
effusion   (previously  sterilised,    if    necessary,    by    the    fractional 
method) ;  allow  the  tubes  to  solidify  in  a  sloping  position. 

6.  When  solid,  incubate  at  37°  C.  for  forty-eight  hours,  and 
eliminate    any    contaminated    tubes.     Store    the    remainder    for 
future  use. 

Serum  Agar  (Wertheimer).— 

1.  Prepare   nutrient    agar    (vide   page    167),    to    the    following 
formula : 

Agar 2.0  per  cent. 

Peptone 2.0  per  cent. 

Salt 0.5  per   cent. 

Meat  extract quantum  sufficit. 

2.  Make  reaction  of  medium  +  10. 

3.  Filter;  tube  in  quantities  of  5  c.c. 

4.  Sterilise  as  for  nutrient  agar. 

5.  After  the  last  sterilisation  cool  to  42°  C.,  then  add  5  c.c. 
sterile  blood-serum  from  human  placenta  (sterilised,  if  necessary, 
by  the  fractional  method)  to  each  tube;  slope  the  tubes. 

6.  When   solid,   incubate   at   37°  C.  for  forty-eight  hours,  and 
eliminate    any    contaminated    tubes.     Store    the    remainder    for 
future  use. 

Serum  Agar  (Kanthack  and  Stevens). — 

1.  Collect  ascitic,  pleuritic,  or  hydrocele  fluid  in  sterile  flasks 
and  allow  to  stand  in  the  ice-chest  for  twelve  hours  to  sediment. 

2.  Decant  1000  c.c.  of  the  clear  fluid  into  a  measuring  cylinder 
and  transfer  to  sterile  litre  flask. 

3.  Add  0.5  c.c.  dekanormal  NaOH  solution  for  every  100  c.c. 
serum  (i.e.,  5.0  c.c.),  and  mix  thoroughly. 

4.  Heat  in  the  steamer  for  twenty  minutes. 

5.  Weigh  out  15  grammes  agar,  emulsify  in  a  separate  vessel 
with    200  c.c.  of   the  alkaline  fluid  previously   cooled   to   about 
20°  C.,  and  then  add  to  the  remainder  of  the  fluid  in  the  flask. 

6.  Bubble  live  steam  through  the  mixture  for  twenty  minutes 
to  dissolve  the  agar. 

7.  Filter  through  papier  Chardin,  using  a  hot-water  funnel. 

8.  Weigh  out  glucose  10  grammes  (=i  per  cent.),  and  dissolve 
it  in  the  clear  agar. 

8a.   If  desired,  add  glycerine,  5  per  cent.,  to  the  clear  agar. 

9.  Tube,  and  sterilise  as  for  nutrient  agar. 


212  SPECIAL   MEDIA 

Serum    Agar    (Libman).— 

1.  Prepare  nutrient  agar  (vide,  page  167)  using,  however,   1.5 
per  cent,  peptone    (that  is   15   grammes  per  litre  instead   of   10 
grammes) . 

2.  Adjust  the  reaction  to  o  (i.  e.,  neutral  to  phenolphthalein) . 

3.  Filter  and  transfer   1000  c.c.  liquefied  medium  to  a  sterile 
flask. 

4.  Weigh  out  dextrose  20  grammes  and  dissolve  in  the  fluid 
agar. 

5.  Tube  in  quantities  of  6  c.c.;  and  sterilise  in  the  steamer  at 
1 00°  C.  for  thirty  minutes  on  each  of  three  consecutive  days. 

6.  After  the  third  sterilisation  cool  to  42°  C.  and  add  to  each 
tube  3  c.c.  of  sterile  hydrocele  fluid,  ascitic  fluid  or  pleuritic  effu- 
sion (previously  sterilised,  if  necessary,  by  the  fractional  method) ; 
allow  the  tubes  to  solidify  in  a  sloping  position. 

7.  When  solid,  incubate  at  37°  C.  for  forty-eight  hours,  and 
eliminate    any    contaminated    tubes.      Store    the    remainder    for 
future  use. 

Egg=albumen,  Inspissated. — 

1.  Break  several  fresh  eggs   (hens',  ducks',  or  turkeys'   eggs), 
and  collect  the  "whites"  in  a  graduated  cylinder,  taking  care  to 
avoid  admixture  with  the  yolks. 

2.  Add    40    per    cent,    distilled    water,    and    incorporate    the 
mixture  thoroughly  by  the  aid  of  an  egg- whisk. 

3.  Weigh  out  0.15  per  cent,   sodium  hydrate  and  dissolve  it 
in   the   fluid    (or   add   the   amount   of   dekanormal   caustic   soda 
solution  calculated  to  yield  the  required  percentage  of  soda  in 
the  total  bulk  of  the  fluid — i.  e.,  0.375  c-c-  of  dekanormal  NaOH 
solution  per  100  c.c.  of  the  mixture). 

30.  Glucose  to  the  extent  of  i  to  2  per  cent,  may  now  be  added, 
if  desired. 

4.  Strain  the  mixture  through  butter  muslin  and  filter  through 
a  porcelain  filter  candle  into  a  sterile  filter  flask. 

5.  Tube,  and  stiffen  at  100°  C.  in  the  serum  inspissator. 

6.  Incubate  at  37°  C.  for  forty-eight  hours  and  eliminate  any 
contaminated  tubes;  store  the  remainder  for  future  use. 

Egg=albumen  (Tarchanoff  and  Kolesnikoff). — 

1.  Place  unbroken  hens'  eggs  in  dekanormal  caustic  soda  solu- 
tion for  ten  days.      (After  this  time  the  white  becomes  firm  like 
gelatine.) 

2.  Carefully  remove  the  shell  and  cut  the  egg  into  fine  slices. 

3.  Wash  for  two  hours  in  running  water. 

4.  Place  the  egg  slices  in  a  large  beaker  and  sterilise  in  the 
steamer  at  100°  C.  for  one  hour. 

5.  Transfer  each  slice  of  egg  by  means  of  a  pair  of  sterilised 
forceps  to  a  Petri  dish  or  large  capsule. 


ASCITIC    FLUID   AGAR  213 

6.  Sterilise  in  the  steamer  at  100°  C.  for  twenty  minutes  on 
each  of  three  consecutive  days. 

Egg  Albumin  Broth  (Lipschuetz). — 

1.  Weigh  out 

Egg  albumin  (extra  fine  powder,  Merck) .    .    4  grammes 

and  place  in  a  2 -litre  flask  with  a  number  of  sterile  glass  beads. 

2.  Measure  out  distilled  water  200  c.c.  into  a  half -litre  flask 
and  warm  to  37°  C.  in  the  incubator. 

3.  Add  the  water  to  the  flask  containing  the  albumin  and  beads 
and  dissolve  by  shaking. 

4.  Add    *Q  NaOH,    40  c.c.     Allow   the   mixture    to    stand  for 
thirty  minutes  with  frequent  shaking. 

5.  Filter  through  Swedish  filter  paper. 

6.  Sterilise  by  boiling  two  or  three  times  at  intervals  of  two 
hours. 

7.  Add  ordinary  nutrient  bouillon  600  c.c. 

8.  Fill  into  small  Erlenmeyer  flasks  in  quantities  of  50  c.c. 

9.  Incubate  for  forty-eight  hours  at  37°  C. — discard  any  con- 
taminated flasks  and  store  the  remainder  for  future  use. 

Egg  Albumin  Agar.— 

1.  Prepare  egg  albumin  solution  as  above  1-6. 

2.  Liquefy  and  measure  out  ordinary  nutrient  agar  600  c.c.  and 
add  to  the  egg  albumin  solution  (in  place  of  the  nutrient  broth). 

3.  Complete  as  above  8-9. 

Diplococcus  Meningitidis  Intracellularis. 

Ascitic  Fluid  Agar  (Wassermann)  Synonym  N=as=gar  (Mervyn 
Gordon) . 

1.  Liquefy  and  measure  out  into  a  sterile  flask: 

Nutrient  agar 600  c.c. 

2.  Measure  out  into  a  half  litre  flask 

Distilled  water 210  c.c. 

and  add  to  it 

Ascitic  fluid 90  c.c. 

Nutrose 6  grammes 

3.  Heat  over  a  bunsen  flame,  shaking  constantly  until  the  fluid 
boils,  and  the  nutrose  is  dissolved, 

4.  Add  the  nutrose  ascitic  solution  to  the  fluid  agar. 

5.  Heat  in  the  steamer  for  thirty  minutes,  then  filter. 

6.  Tube  and  sterilise  as  for  nutrient  agar. 

NOTE. — The  finished  medium  in  this  case  measures  900  c.c.  only 
since  inconvenient  fractions  would  be  introduced  in  making  up  to 
one  litre  exactly. 


214  SPECIAL   MEDIA 

Diplococcus  Pneumonias. 

Blood  Agar  (Washbourn).— 

1.  Melt  up  several  tubes  of  nutrient  agar  (vide  page  167)  and 
allow  them  to  solidify  in  the  oblique  position. 

2.  Place  the  tubes,  in  the  horizontal  position,  in  the   "hot" 
incubator  for  forty-eight  hours,   to   evaporate   off   some   of  the 
condensation  water. 

3.  Kill  a  small  rabbit  with  chloroform  and  nail  it  out  on  a 
board  (as  for  a  necropsy).     Moisten  the  hair  thoroughly  with  2 
per  cent,  solution  of  lysol. 

4.  Sterilise  several  pairs  of  forceps,  scissors,  etc.,  by  boiling. 

5.  Reflect  the  skin  over  the  thorax  with  sterile  instruments. 

6.  Open  the  thoracic  cavity  by  the  aid  of  a  fresh  set  of  sterile 
instruments. 

7 .  Open  the  pericardium  with  another  set  of  sterile  instruments. 

8.  Sear  the  surface  of  the  left  ventricle  with  a  red-hot  iron 
and  remove  fluid  blood  from  the  heart  by  means  of  sterile  pipettes 
(e.  g.,  those  shown  in  Fig.  13,  c}. 

9.  Deliver  a  small  quantity  of  the  blood  on  the  slanted  surface 
of  the  agar  in  each  of  the  tubes,  and  allow  it  to  run  over  the 
entire  surface  of  the  medium. 

10.  Place  the  tubes  in    the    slanting   position    and    allow    the 
blood  to  coagulate. 

11.  Return  the  "blood  agar"  to  the  hot  incubator  for  forty- 
eight  hours  and  eliminate  any  contaminated  tubes.     Store  the 
remainder  for  future  use. 

Media  for  the  Study  of  Mouth  Bacteria  Generally. 

Potato  Gelatine  (Goadby).— 

1.  Prepare  glycerine  potato  broth  (see  page  203,  sections  i  to  5). 

2.  Add    10    per   cent,    gelatine   to    the    potato   decoction    and 
bubble  live  steam  through  the  mixture  for  ten  minutes. 

3.  Estimate  the  reaction;  adjust  the  reaction  of  the  medium 
to  +5. 

4.  Cool  the  medium  to  below  60°  C.,  clarify  with  egg  as  for 
nutrient  gelatine. 

5.  Filter  through  papier  Chardin. 

6.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

Media  for  the  Study  of  Protozoa. 

Tissue  Medium  (Noguchi). — For  spirochates  (cultivations  must  be 
grown  anaerobically} . 

1.  Plug  and  sterilise  test-tubes  20 X  2  cm. 

2.  Kill  a  small  rabbit  with  chloroform  vapour.     Open  the  abdo- 


TISSUE    MEDIUM  215 

men  with  all  aseptic  precautions,  remove  kidneys  and  testicles  and 
transfer  to  a  sterile  glass  dish.  Cut  up  the  organs  with  sterile 
scissors  into  small  pieces — say  4  millimetre  cubes.  The  four  organs 
should  yield  from  25  to  30  pieces  of  tissue. 

3.  Drop  a  small  piece  of  sterile  tissue  into  the  bottom  of  each 
sterilised  tube. 

4.  Take  a  flask  containing  about  400  c.c.  nutrient  agar  (+  10 
reaction) ,  liquefy  the  medium  by  heat  and  cool  in  a  water  bath 
to  50°  C. 

5.  Add  200  c.c.  ascitic  or  hydrocele  fluid  (horse  or  sheep  serum 
may  be  employed,  but  is  not  so  good)  to  the  liquid  agar  and  mix 
carefully  to  avoid  formation  of  air  bubbles. 

6.  Fill  about  20  c.c.  of  the  ascitic  agar  into  each  of  the  sterilised 
tubes  which  already  contains  a  piece  of  sterile  rabbit's  tissue,  stand 
all  the  tubes  upright  in  racks  or  a  jar,  and  allow  agar  to  set. 

7.  After  solidification  pour  sterile  paraffin  oil  on  the  surface  of 
the  medium  in  each  tube  to  the  depth  of  3  centimetres. 

8.  Incubate  tubes  at  37°  C.  for  several  days  and  discard  any 
which  prove  to  be  contaminated. 

9.  Store  such  tubes  as  are  sterile  for  future  use. 


XIII.  INCUBATORS. 

AN  incubator  (Fig.  113)  consists  essentially  of  a  cham- 
ber for  the  reception  of  cultivations,  etc.,  surrounded  by 
a  water  jacket,  the  walls  of  which  are  of  metal,  usually 
copper,  and  outside  all  an  asbestos  or  felt  jacket,  or 


FIG.  113. — Incubator- 

wooden  casing.  The  water  in  the  jacket  is  heated 
by  gas  or  electricity  and  maintained  at  some  constant 
temperature  by  a  thermo-regulator.  The  cellular 
incubator  (Fig.  114)  which  was  made  for  me1  some 
years  ago  is  of  the  greatest  practical  utility.  Here  the 

1  Made  by  the  firm  of  Chas.  Hearson  &  Co.,  235  Regent  St.,  London,  W. 

2l6 


THERMO-REGULATORS 


217 


central  cavity  is  subdivided  by  five  double  -walled 
partitions  (in  which  water  circulates  in  connection  with 
the  water  tanks  at  the  top  and  base  of  the  incubator) 
and  again  by  iron  shelves  to  form  twenty-four  pigeon 
holes.  Into  each  of  these  slides  an  iron  drawer  35  cm. 
long  X  12  cm.  wide  X  22  cm.  high  forming  a  self- 
contained  incubator.  The  drawer  is  fitted  with  a 
wooden  form  to  which  is  fixed  a  handle  and  a  numbered 
label.  The  thermo-regulating  apparatus  is  the  well- 
known  Hearson  capsule. 


Fio.  114. — Cellular  incubator. 

Two  incubators  at  least  are  required  in  the  labora- 
tory, for  the  cultivation  of  bacteria  the  one  regulated 
to  maintain  a  temperature  of  37°  C.,  and  known  as 
the  "hot"  incubator;  the  other,  20°  C.  to  22°  C.,  and 
known  as  the  "cool"  or  "cold"  incubator. 

Two  other  incubators,  regulated  to  42°  C.  and  60°  C. 
respectively,  whilst  not  absolutely,  necessary  very  soon 
justify  their  purchase. 

Thermo=regulators. — The    thermo-regulator    is    the 


2l8  INCUBATORS 

most  essential  portion  of  the  incubator,  as  upon  its 
efficient  working  depends  the  maintenance  of  a  con- 
stant temperature  in  the  cultivation  chamber.  It  is 
also  used  in  the  fitting  up  of  water  and  paraffin  baths, 
and  for  many  other  purposes. 

Of  the  many  forms  and  varieties  of  thermo-regulator 
(other  than  electrical),  two  only  are  of  sufficiently 
general  use  to  need  mention.  In  one  of  these  the  flow 
of  gas  to  the  gas-jet  is  controlled  by  the  expansion  or 
contraction  of  mercury  within  a  glass 
bulb ;  in  the  other,  by  alterations  in 
the  position  of  the  walls  of  a  metal- 
lic capsule  containing  a  fluid,  the 
boiling-point  of  which  corresponds 
to  the  temperature  at  which  the  in 
cubator  is  intended  to  act.  They 
are: 

(a)  Reichert's  (Fig.  115),  consists  of 
a  bulb  containing  mercury  which  is 
FIG.  1.15.— Rekhert's  to    be    suspended    in    the   medium, 
thermo-regulator.     whether  air  or  waterj  the  temperature 

of  which  it  is  desired  to  regulate.  Gas  enters  at  A,  and 
passes  out  to  the  jet  by  B.  As  the  temperature  rises 
the  mercury  expands  and  cuts  off  the  main  gas  supply. 
As  the  temperature  falls  the  mercury  contracts  and  re- 
opens the  narrow  tube  C.  By  means  of  a  thumbscrew 
D  (which  mechanically  raises  or  lowers  the  column  of 
mercury  irrespective  of  the  temperature)  and  the  aid 
of  a  thermometer  the  apparatus  can  be  set  to  keep  the 
incubator  at  any  desired  temperature.  With  this  form 
a  special  gas  burner  is  required,  with  separate  supply  of 
gas  to  a  pilot  jet  at  the  side. 

(b)  Hear  son's  capsule  regulator  consists  of  a  metal  cap- 
sule hermetically  sealed  and  filled  with  a  liquid  which 
boils  at  the  required  temperature,  this  is  adjusted  in  the 
interior  of  the  incubator.  Soldered  to  the  upper  side  of 
the  capsule  is  a  thick  piece  of  metal  having  a  central 


THERMO-REGULATORS  2IQ 

cup  to  receive  the  lower  end  of  a  rigid  rod,  through 
which  the  movements  of  the  walls  of  the  capsule  are 
transmitted  to  the  gas  valve  fixed  outside  the  incubator. 

The  gas  valve  or  governor  is  shown  in  figure  1 16.  A 
is  the  inlet  for  gas,  C  the  outlet  to  burner  heating  the 
water  jacket,  B  D  a  lever  pivoted  to  standards  at  G, 
and  acted  upon  by  the  capsule,  through  the  rigid  rod 
which  enters  the  socket  below  the  screw  P. 

The  construction  of  the  valve  is  such  that,  when- 
ever the  short  arm  of  the  lever  B  D  presses  on  the  disc 
below  the  end  B,  the  main  supply  of  gas  is  entirely  cut 
off.  At  such  times,  however,  a  very  small  portion  of 
gas  passes  from  A  to  C,  through  an  aperture  inside  the 
valve,  the  size  of  which  aperture  can  be  adjusted  by 


FIG.  116. — Capsule  thermo-regulator. 

the  screw  needle  S,  hence  the  gas  flame  below  the  incu- 
bator is  never  extinguished. 

The  expansion  of  the  metal  walls  of  the  capsule, 
which  takes  place  upon  the  boiling  of  its  contents,  pro- 
vides the  motive  force,  transmitted  through  the  rigid 
rod  to  raise  the  long  arm  of  the  lever  B  D,  and  as  this 
expansion  only  takes  place  at  a  predetermined  temper- 
ature, the  lever  will  only  be  acted  upon  when  the  criti- 
cal temperature  is  reached,  no  sensible  effect  being  pro- 
duced at  even  i°  C.  below  that  at  which  the  capsule  is 
destined  to  act. 

W  is  a  weight  sliding  on  the  lever  rod  D;  by  increas- 
ing the  distance  between  the  weight  and  the  fulcrum 


220  INCUBATORS 

of  the  lower  increased  pressure  is  brought  to  bear  upon 
the  walls  of  the  capsule  with  the  result  that  the  boiling- 
point  of  the  liquid  in  the  capsule  is  slightly  raised,  and 
a  range  of  about  two  degrees  can  thus  be  obtained 
with  any  particular  capsule. 


XIV.  METHODS  OF  CULTIVATION. 

CULTIVATIONS  of  micro-organisms  are  usually  pre- 
pared in  the  laboratory  in  one  of  three  ways : 

Tube  cultures. 
Plate  cultures. 
Hanging=drop  cultures. 

These  may  be  incubated  either  aerobically  (/.  e., 
in  the  presence  of  oxygen)  or  anaerobically  (i.  e.,  in 

the  absence  of  oxygen,  or  in  the  presence  of  an  in- 
different gas,  such  as  hydrogen,  nitrogen,  or  carbon 
dioxide) . 

With  regard  to  the  temperature  at  which  the  culti- 
vations are  grown,  it  may  be  stated  as  a  general  rule 
that  all  media  rendered  solid  by  the  addition  of  gela- 
tine are  incubated  at  20°  C.,  or  at  any  rate  at  a  tem- 
perature not  exceeding  22°  C.  (that  is,  in  the  "cold" 
incubator) ;  whilst  fluid  media  and  all  other  solid  media 
are  incubated  at  3  7°  C.  (that  is,  in  the ' '  hot "  incubator) . 
Exceptions  to  this  rule  are  numerous.  For  instance, 
in  studying  the  growth  of  the  psychrophylic  bacteria, 
the  yeasts  and  the  moulds,  the  cold  incubator  is  em- 
ployed for  all  media. 

Tube  cultivations  are  usually  packed  in  the  incubator 
in  small  tin  cylinders,  such  as  those  in  which  American 
cigarettes  are  sold,  or  in  square  tin  boxes.  Beakers  or 
tumblers  may  be  used  for  the  same  purpose,  but  being 
fragile  are  not  so  convenient.  Metal  test-tube  racks, 
long  enough  to  just  fit  into  the  interior  of  the  incuba- 
tor and  each  accommodating  two  rows  of  tubes,  are 
also  exceedingly  useful. 

221 


222  METHODS    OF   CULTIVATION 

AEROBIC. 

The  Preparation  of  Tube  Cultivations. 

The  preparation  of  a  tube  cultivation  consists  in: 

(a)  Inoculating  a  tube  of  sterile  nutrient  medium 
with  a  portion  of  the  material  to  be  examined. 

(b)  Incubating  the  inoculated  tube  at  a  suitable 
temperature. 

The  details  of  the  first  of  these  processes  must  be 
varied  somewhat  according  to  whether  the  tubes  of 
nutrient  media  are  inoculated  or  "planted"  from — 

1.  Pre-existing  cultivations. 

2.  Morbid  material  previously  collected  (vide  page 

373)- 

3 .  Fluids,  tissues,  etc. ,  or  from  the  animal  body  direct. 
The  method   of  preparing  tube   cultivations   from 

pre-existing  cultivations  is  as  follows : 

1.  Fluid  Media  (e.  g.,  Nutrient  Bouillon).— 

i .  Flame  the  cotton-wool  plug  of  the  tube  containing 


FIG.  117. — Inoculating  tubes,  seen  from  the  front. 

the  cultivation  and  also  that  of  the  tube  of  sterile 
bouillon. 

2.  Hold  the  two  tubes,  side  by  side,  between  the 
left  thumb  and  the  first  and  third  ringers,  allowing  the 
sealed  ends  to  rest  on  the  dorsum  of  the  hand,  and 
separating  the  mouths  of  the  tubes  (which  are  pointed 
to  the  right)  by  the  tip  of  the  second  finger.  Keep 


SOLID    MEDIA,  223 

the  tubes  as  nearly  horizontal  as  is  possible  without 
allowing  the  fluid  in  the  bouillon  tube  to  reach  the 
cotton-wool  plug  (Fig.  117).  . 

3.  Sterilise  the  platinum  loop  and  allow  it  to  cool.1 

4.  Grasp  the  plug  of  the  tube  containing  the  culti- 
vation between  the  little  finger  and  palm  of  the  hand 
and  remove  it  from  the  tube. 

5.  Grasp  the  plug  of  the  bouillon  tube  between  the 
fourth  finger  and  the  ball  of  the  thumb  and  remove  it 
from  the  tube. 

6.  Pass  the  platinum  loop  into  the  tube  containing 
the  culture — do  not  allow  the  loop  to  touch  the  sides 
of  the  tube,  or  the  handle  to  touch  the  medium — and 
remove  a  small  portion  of  the  growth;  withdraw  the 
loop  from  the  tube,  keeping  the  infected  side  of  the 
loop  downward. 

7.  Pass  the  loop  into  the  bouillon  tube  almost  down 
to  the  level  of  the  fluid,  reverse  the  loop  so  that  the 
infected  side  faces  upward,  emulsify  the   portion   of 
the  growth  in  the  moisture  adhering  to  the  side  of  the 
tube  which  is  uppermost.     Withdraw  the  loop. 

8.  Replug  both  tubes. 

9.  Sterilise  the  platinum  loop. 

10.  Label  the  bouillon  tube  with  (a)  the  name  of 
the  organism  and  (b)  the  date  of  inoculation. 

11.  Incubate. 

2.  Solid  Media. — Solid  media  are  stored  in  tubes  in 
one  of  two  ways : 

1.  Oblique  tube  or  slanted  tube  (Fig.  118),  in  which 
the  medium  has  been  allowed  to  solidify  whilst  the  tube 
was  retained  in  an  inclined  position,  so  forming  an 
extensive  surface  of  medium  extending  from  the  bot- 
tom of  the  tube  almost  to  its  mouth. 

This  is  employed  for  " streak"  or  " smear"  cultiva- 
tions (Strichcultur) . 

2.  Straight  tube  (Fig.   119),  in  which  the  medium 

1  See  also  method  of  opening  and  closing  culture  tubes,  pages  74-7°- 


224  METHODS    OF   CULTIVATION 

forms  a  cylindrical  mass  in  the  lower  portion  of  the 
tube  and  presents  an  upper  surface  which  is  at  right 
angles  to  the  long  axis  of  the  tube. 

This  is  employed  for  "stab"  or  " stick"  cultivations 
(Stichcultur) ,  or  by  inoculating  the  medium  whilst 
fluid,  and  allowing  to  solidify  in  this  position,  for 
"shake"  cultivations. 

Streak  Culture. — 

1.  Flame  the  plugs,  sterilise  the  platinum  loop  (or 
spatula).     Open  the  tubes  and  charge  the  loop  as  in 
previous  inoculation. 

2.  Pass  the  infected  loop  to  the  bottom  of  the  tube 
to  be  inoculated  and  draw  it,  as  lightly  as  possible, 
along  the  centre  of  the  surface  of  the  medium,  ter- 
minating the  " streak"  over  the  thin  layer  of  medium 
near  the  mouth  of  the  tube. 

3.  Replug  the  tubes,   sterilise  the  platinum  loop. 

4.  Label  the  newly  inoculated  tube  and  incubate. 

Smear  Culture. — Proceed  generally  as  in  streak  cul- 
ture, but  rub  the  infected  loop  all  over  the  surface  of 
the  medium,  instead  of  restricting  the  inoculation  to 
a  narrow  line. 

NOTE. — Gelatine  and  agar  oblique  tubes  should  be  freshly 
"slanted"  before  use. 

Stab  Culture. — 

1.  Flame  the  plugs,   open  the  tubes,   sterilise  the 
platinum  needle  and  charge  it  with  the  inoculum  as 
in  the  previous  cultivations. 

2.  Pass  the  platinum  needle  into  the  tube  to  be 
inoculated  until  it  touches  the  centre  of  the  surface  of 
the   medium.     Now  thrust   it   deeply  into   the   sub- 
stance of  the  medium,  keeping  the  needle  as  nearly  as 
possible  in  the  axis  of  the  cylinder  of  medium.     Then 
withdraw  the  needle. 

3.  Replug  the  tubes.     Sterilise  the  platinum  needle. 


TUBE    CULTURES 


225 


4.  Label   the   newly   planted   tube   and   incubate. 

NOTE. — When  gelatine  is  stored  for  some  time  the  upper 
surface  of  the  cylinder  becomes  concave  owing  to  evaporation. 
Tubes  showing  this  appearance  should  be  liquefied  and  again 
allowed  to  set  before  use  for  stab  culture,  otherwise  when  the 
needle  enters  the  medium,  the  surface  tension  will  cause  the 
gelatine  cylinder  to  split. 


FIG.  118. — Sloped  or  slant- 
ed medium  for  streak  or 
smear  culture. 


FIG.  119. — Straight  tube. 


Shake  Culture. — 

1.  Liquefy  a  tube  of  nutrient  gelatine  (or  agar,  or 
other  similar  medium),   by  heating  in  a  water-bath 
(Fig.  1 21). 

2.  Inoculate  the  liquefied  medium  and  label  it,  etc., 
precisely  as  if  dealing  with  a  tube  of  bouillon. 


226 


METHODS    OF   CULTIVATION 


3.  Place   the   newly   planted   tube   in   the   upright 
position   (e.  g.,  in  a  test-tube  rack)   and  allow  it  to 
solidify. 

4.  Label  the  tube;  when  solid,  incubate. 

Esmarch's  Roll  Cultivation. — 

1.  Liquefy  three  tubes  of  gelatine  by  heat. 

2.  Prepare  three  dilutions  of  the  inoculum   (as  described  for 
plate  cultivations,  page  228,  steps  4  to  7). 

3.  Roll  the  tubes,  held  almost  horizontally,  in  a  groove  made 
in  a  block  of  ice,  until  the  gelatine  has  set  in  a  thin  film  on  the 
inner  surface  of  tube  (Fig.  120) ;  or  under  the  cold-water  tap. 


FIG.  120. — Esmarch's  roll  culture  on  block  of  ice. 

In  order  that  the  medium  may  adhere  firmly  to  the  glass,  the 
agar  used  for  roll  cultivation  should  have  i  per  cent,  gelatine  or 
i  per  cent,  gum  arabic  added  to  it  before  sterilisation. 

Roll  cultivations,  which  served  a  most  important  purpose  in 
the  days  before  the  introduction  of  Petri  dishes  for  plate 
cultivations,  are  now  obsolete  in  modern  laboratories  and  are 
merely  mentioned  for  the  benefit  of  students,  since  examiners  who 
are  interested  in  the  academic  and  historical  aspects  of  bacteriology 
sometimes  expect  candidates  to  be  acquainted  with  the  method  of 
preparing  them. 

The  Preparation  of  Plate  Cultures. 

If  a  small  number  of  bacteria  are  suspended  in  lique- 
fied gelatine,  agar,  or  other  similar  medium,  and  the  in- 
fected medium  spread  out  in  an  even  layer  over  a  flat 
surface  and  allowed  to  solidify,  each  individual  micro- 
organism becomes  fixed  to  a  certain  spot  and  its 
further  development  is  restricted  to  the  vicinity  of  this 
spot.  After  a  variable  interval  the  growth  of  this 


POURING"    PLATES 


227 


organism  becomes  visible  to  the  naked  eye  as  a  "  colony." 
This  is  the  principle  upon  which  the  method  of  plate  cul- 
tivation is  based  and  its  practice  enables  the  bacteri- 
ologist to  study  the  particular  manner  of  development 
affected  by  each  species  of  microbe  when  growing 
(a)  unrestricted  upon  the  sur- 
face of  the  medium,  (b)  in  the 
depths  of  the  medium.  The 
method  itself  is  as  follows: 

Apparatus  Required.  — 

1.  Tripod  levelling  stand. 

2.  Large  shallow  glass  dish,  with  a 
square  sheet  of  plate  glass  to  cover  it. 

3.  Spirit  level. 

4.  Case  of  sterile  Petri  dishes. 

5.  Tubes  of  sterile  nutrient  media, 
gelatine  (or  agar)  previously  liquefied 
by   heating    in    the   water-bath   and 
cooled  to  42°   C.,  otherwise  the  heat 
of  the  medium  would  destroy  many, 
if  not  all,  of  the  bacteria  introduced. 

6.  Tube  of  cultivation  to  be  planted 
from. 

7.  Platinum  loop. 

8.  Bunsen  burner. 

9.  Grease  pencil. 

FIG.    121.  —  Handy    form   of 


Methodof  "Pouring"  Plates.- 

1.  Place  the  glass  dish  on  the 
levelling  tripod  (Figs.  122,  123);  plates 

if  gelatine  plates  are  to  be  poured  fill  the  dish  with 
ice  water  —  gelatine  solidifies  so  slowly  that  it  is 
necessary  to  hasten  the  process  ;  if  agar  is  to  be  used 
fill  with  water  at  50°  C.  —  agar  sets  almost  immediately 
at  the  room  temperature  and  by  slightly  retarding  the 
process  lumpiness  is  avoided  ;  cover  the  dish  with  the 
square  sheet  of  glass. 

2.  Place  the  spirit  level  on  the  sheet  of  glass  and  by 
means  of  the  levelling  screws  adjust  the  surface  of  the 
glass  to  the  horizontal. 


228 


METHODS   OF  CULTIVATION 


This  leveling  is  an  important  matter  since  the  de- 
velopment of  a  colony  is  to  some  extent  proportionate 
to  the  supply  of  medium  available  for  its  nutrition. 
Thus  in  a  "smear"  on  sloped  tube  culture,  the  colonies 
at  the  upper  part  of  the  medium  are  stunted  and  small 


FIG.  122. — Plate-levelling  stand. 

but  increase  in  size  and  luxuriance  of  growth  the 
nearer  they  approach  to  the  bottom  of  the  tube, 
where  there  is  the  greatest  depth  of  medium. 

3.  Place  three  sterile  Petri  dishes  in  a  row  on  the 
surface  of  the  glass  plate  and  number  them  i,  2,  and 
3,  from  left  to  right. 


FIG.  123. — Plate-levelling  stand,  side  view. 

4.  Number  the  previously  liquefied  tubes  of  nutrient 
media  1,2,  and  3.     Flame  the  plugs  and  see  that  each 
plug  can  be  readily  removed  from  the  mouth  of  its 
tube. 

5.  Add  one  loopful  of  the  inoculum  to  tube  No.  i, 


POURING       PLATES 


229 


treating  the  liquefied  medium  as  bouillon.  After  re- 
plugging, grasp  the  tube  near  its  mouth  by  the  thumb 
and  first  ringer  of  the  right  hand,  and  with  an  even 
circular  movement  of  the  whole  arm,  diffuse  the  inocu- 
lum throughout  the  medium ;  avoid  jerky  movements, 
as  these  cause  bubbles  of  air  to  form  in  the  medium. 


FIG.  124. — Mixing  emulsion  for  plates. 

The  knack  of  mixing  evenly  without  producing  air 
bubbles,  is  not  always  easily  acquired,  by  this  method, 
An  alternative  plan  is  to  hold  the  inoculated  tube 
vertically  upright  between  the  opposed  palms  and  to 
rotate  it  between  them  by  rapid  backward  and  forward 
movements  of  the  two  hands  (Fig.  124). 


FIG.  125. — Pouring  plates. 

6.  Sterilise  the  platinum  loop,  and  add  two  loopfuls 
of  diluted  inoculum  to  tube  No.  2,  and  mix  as  before. 

7.  In  a  similar  manner  transfer  three  loopfuls  of 
liquefied  medium  from  tube  No.  2  to  tube  No.  3,  and 
mix  thoroughly. 

8.  Flame  the  plug  of  tube  No.  i,  remove  it,  then  flame 
the  lips  of  the  tube;  slightly " raise  the  cover  of  Petri 
dish  No.   i,  introduce  the  mouth  of  the  tube;  then, 


230  METHODS    OF    CULTIVATION 

elevating  the  bottom  of  the  tube,  pour  the  liquefied 
medium  into  the  Petri  dish,  to  form  a  thin  layer. 
Remove  the  mouth  of  the  tube  and  close  the " plate." 
If  the  medium  has  failed  to  flow  evenly  over  the  bottom 
of  the  plate,  raise  the  plate  from  the  levelling  platform 
and  by  tilting  in  different  directions  rectify  the  fault. 

9.  Pour  plates  No.  2  and  No.  3,  in  a  similar  manner, 
from  tubes  Nos.  2  and  3. 

10.  Label  the  plates  with  the  distinctive  name  or 
number  of  the  inoculum,  also  the  date;  the  number  of 
the  dilution  having  been  previously  indicated  (step  3). 

11.  Place  in  the  cool  incubator  for  three  or  more 
days,  as  may  be  necessary. 

In  this  way  colonies  may  be  obtained  quite  pure  and 
separate  from  each  other. 

In  plate  No.  i,  probably,  the  colonies  will  be  so 
numerous  and  crowded,  and  therefore  so  small,  as  to 
render  it  useless.  In  plate  No.  2  they  will  be  more 
widely  separated,  but  usually  No.  3  is  the  plate  reserved 
for  careful  examination,  as  in  this  the  colonies  are  usu- 
ally widely  separated,  few  in  number,  and  large  in  size. 

Agar  plates  are  poured  in  a  similar  manner,  but  the 
agar  must  be  melted  in  boiling  water  and  then  allowed 
to  cool  t045°C.  or42°C.  in  a  carefully  regulated  water- 
bath  before  being  inoculated,  and  the  entire  process 
must  be  carried  out  very  rapidly,  otherwise  the  agar 
will  have  solidified  before  the  operation  is  completed. 

NOTE. — In  pouring  plates,  since  tube  No.  i  (for  the  first  dilu- 
tion) rarely  gives  a  plate  that  is  of  any  practical  value  it  is  fre- 
quently replaced  by  a  tube  of  bouillon  or  sterile  salt  solution,  and 
in  such  case  plate  No.  i  is  not  poured. 

Surface  Plates.— 

This  method  of  pouring  what  may  be  termed  "  whole  " 
plates  (since  colonies  may  appear  both  on  the  surface 
and  in  the  depths  of  the  medium)  is  essential  to  the 
accurate  study  of  the  formation  of  colonies  under 


SURFACE    PLATES  23  F 

various  conditions,  but  when  the  main  object  of  the 
separation  of  the  bacteria  is  to  obtain  subcultivations 
from  a  number  of  individual  bacteria,  "surface"  plates 
must  be  prepared,  since  here  colony  formation  is 
restricted  to  the  surface  of  the  medium.  The  method 
adopted  varies  slightly  according  to  whether  the 
medium  employed  is  gelatine  or  agar,  or  one  of  the  de- 
rivatives or  variants  of  the  latter. 

(a)  Gelatine  Surface  Plates.— 

1.  Liquefy  three  tubes  of  nutrient  gelatine. 

2.  Pour  each  tube  into  a  separate  Petri  dish  and  al- 
low it  to  solidify.     Then  turn  each  plate  and  its  cover 
upside  down. 

3.  When     quite    cold    . 
raise  the  bottom  of  plate 

i,  revert  it  and  deposit  a 
drop    of    the    inoculum 

,,    .  ..         ,  FIG.  126. — Surface  plate  spreader. 

(whether  a  fluid  culture 

or  an  emulsion  from  solid  culture)  upon  the  surface 
of  the  gelatine  with  a  platinum  loop — close  to  one  side 
of  the  plate ;  replace  the  bottom  half  of  the  Petri  dish 
in  its  cover. 

4.  Take  a  piece  of  thin  glass  rod,  stout  platinum  wire 
or  best  of  all  a  piece  of  aluminium  wire  (say  2  mm. 
diameter)  about  28  cm.  long.     Bend  the  terminal  4  cm. 
at  right  angles  to  the  remainder,  making  an  L-shaped 
rod  (Fig.  126).     Sterilise  the  short  arm  and  adjacent 
portion  of  the  long  arm,  in  the  Bunsen  flame,  and  allow 
it  to  cool. 

5.  Now  raise  the  bottom  of  the  Petri  dish  in  the  left 
hand,  leaving  the  cover  on  the  laboratory  bench,  and 
holding  it  vertically,  smear  the  drop  of  inoculum  all 
over  the  surface  of  the  gelatine  with  the  short  arm  of 
the  spreader  by  a  rotatory  motion,  (Fig.  127).     Replace 
the  dish  in  its  cover. 

6.  Raise  the  bottom  of  plate  2  and  rub  the  infected 


232  METHODS    OF   CULTIVATION 

spreader  all  over  the  surface  of  the  gelatine — then  go 
on  in  like  manner  to  the  third  plate  in  the  series. 

7.  Sterilise  the  spreader. 

8.  Label  and  incubate  the  plates. 

After  incubation,  plate  No.  i  will  probably  yield  an 
enormous  number  of  colonies;  plate  2  will  show  fewer 
colonies,  since  only  those  bacteria  adhering  to  the  rod 


FIG.  127. — Spreading  surface  plate. 

after  rubbing  over  plate  i  would  be  deposited  on  its 
surface,  and  by  the  time  the  rod  reached  plate  3  but 
very  few  organisms  should  remain  upon  it.  So  that 
the  third  plate  as  a  rule  will  only  show  a  very  few 
scattered  colonies,  eminently  suitable  for  detailed 
study. 

(b)  Agar  Surface  Plates. — 

1.  Liquefy  three  tubes  of  nutrient  agar — nutrose 
agar  or  the  like. 

2 .  Pour  each  tube  into  a  separate  Petri  dish  and  allow 
it  to  solidify. 

3 .  When  quite  solid  invert  each  dish,  raise  the  bottom 
half  and  rest  it  obliquely  on  its  inverted  cover  (Fig.  128) 
and  place  it  in  this  position  in  an  incubator  at  60°  C. 
for  forty-five  minutes  (or  in  an  incubator  at  42°  C.  for 


HANGING-DROP    CULTURES  233 

two  hours) .     This  evaporates  the  water  of  condensation 
and  gives  the  medium  a  firm,  dry  surface. 

4.  On  removing  the  plates  from  the  incubator  close 
each  dish  and  place  it — still  upside 

down — on  the  laboratory  bench. 

5.  Inoculate  the  plates  in  series  of 
three,  as  described  for  gelatine  sur- 

face  plates  3-8.  FlG  ng  sur. 

face  plate  of  agar. 

Hanging=drop  Cultivation. 

Apparatus  Required. — 

Hanging  drop  slides. 

Cover-slips. 

Section  rack  (Fig.  75). 

Blotting  paper. 

Bell  glass  to  cover  slides. 

Original  culture. 

Tubes  of  broth,  or  liquefied  gelatine  or  agar. 

Forceps. 

Platinum  loop. 

Bunsen  burner. 

Grease  pencil. 

Sterile  vaseline. 

Lysol. 

(a)  Fluid  Media.— 

1.  Prepare  first  and  second  dilutions  of  the  inoculum 
as  directed  for  plate  cultivations  (vide  pages  228—229, 
sections  4  to  6) ,  substituting  tubes  of  nutrient  broth 
for  the  liquefied  gelatine. 

2.  Sterilise  a  hanging-drop  slide  by  washing  thor- 
oughly in  water  and  drying,  then  plunging  it  into  a 
beaker  of  absolute  alcohol,   draining  off  the  greater 
part  of  the  spirit,  grasping  the  slide  in  a  pair  of  forceps, 
and  burning  off  the  remainder  of  the  alcohol  in  the 
flame. 

3 .  Place  the  hanging-drop,  slide  on  a  piece  of  blotting 
paper  moistened  with  2  per  cent,  lysol  solution  and 


234  METHODS    OF   CULTIVATION 

cover  it  with  a  small  bell  glass  that  has  been  rinsed 
out  with  the  same  solution  and  not  dried. 

4.  Raise  the  bell  glass  slightly  and  smear  sterile 
vaseline  around  the  rim  of  the  metal  cell  by  means  of  a 
sterile  spatula  of  stout  platinum  wire. 

5.  Remove  a  clean  cover-slip  from  the  alcohol  pot 
with  sterile  forceps  and  burn  off  the  alcohol;  again 
raise  the  bell  glass  and  place  the  sterile  cover-slip  on 
the  blotting  paper  by  the  side  of  the  hanging-drop  slide. 

6.  Remove  a  drop  of  the  broth  from  the  second 
dilution  tube  with  a  large  platinum  loop;  raise  the 
bell  glass  and  deposit  the  drop  on  the  centre  of  the 
cover-slip.     Sterilise  the  loop. 

7.  Raise  the  bell  glass  sufficiently  to  allow  of  the 
cover-slip  being  grasped  with  forceps,  inverted,  and 
adjusted  over  the  cell  of  the  hanging-drop  slide.     Re- 
move the  bell  glass  altogether  and  press  the  cover-slip 
firmly  on  to  the  cell. 

8.  Either  incubate  and  examine  at  definite  intervals, 
or  observe  continuously  with  the  microscope,   using 
a  warm  stage  if  necessary  (Fig.  53). 

(b)  Solid  Media. — Observing  precisely  similar  tech- 
nique, a  few  drops  of  liquefied  gelatine  or  agar  from 
the  second  dilution  tube  may  be  run  over  the  surface 
of  the  sterile  cover-slip  and  a  hanging-drop  plate  cul- 
tivation thereby  prepared. 

This  method  is  extremely  useful  in  connection  with 
the  study  of  yeasts,  when  the  circular  cell  on  the 
hanging-drop  slide  should  be  replaced  by  a  rectangu- 
lar cell  some  38  by  19  mm.,  and  the  gelatine  spread 
over  a  cover-slip  of  similar  size.  After  sealing  down 
the  preparation,  the  upper  surface  of  the  cover-slip 
may  be  ruled  into  squares  by  the  aid  of  the  grease 
pencil  or  a  writing  diamond  and  numbered  to  facilitate 
the  subsequent  identification  of  the  colonies  which  are 
observed  to  develop  from  solitary  germs. 


HANGING-BLOCK    CULTURE  235 

Hanging-block  Culture  (Hill).— 

Apparatus  required:  As  for  hanging-drop  cultivation 
with  the  addition  of  a  scalpel. 

Carry  out.  the  method  as  far  as  possible  under  cover 
of  a  bell  glass,  to  avoid  aerial  contamination. 

1.  Liquefy  a  tube  of  nutrient  agar  (or  gelatine)  and 
pour  into  a  Petri  dish  to  the  depth  of  about  4  mm.  and 
allow  to  set. 

2.  With  a  sharp  scalpel  cut  out  a  block  some  8  mm. 
square,  from  the  entire  thickness  of  the  agar  layer. 

3.  Raise  the  agar  block  on  the  blade  of  the  scalpel 
and  transfer  it,  under  side  down,  to  the  centre  of  a 
sterile  slide. 

4.  Spread  a  drop  of  fluid  cultivation  (or  an  emulsion 
of  growth  from  a  solid  medium)  over  the  upper  surface 
of  the  agar  block  as  if  making  a  cover-slip  film. 

5 .  Place  the  slide  and  block  covered  by  the  bell  glass 
in   the   incubator   at  37°  C.  for  ten  minutes  to  dry 
slightly. 

6.  Take  a  clean  dry  sterile  cover-slip  in  a  pair  of  for- 
ceps, and  with  the  help  of  a  second  pair  of  forceps  lower 
it  carefully  on  the  inoculated  surface  of  the  agar  (avoid- 
ing air  bubbles),  so  as  to  leave  a  clear  margin  of  cover- 
slip  overlapping  the  agar  block. 

7.  Invert  the  preparation  and  with  the  blade  of  the 
scalpel  remove  the  slide  from  the  agar  block. 

8.  With  a  platinum  loop  run  a  drop  or  two  of  melted 
agar  around  the  edges  of  the  block.     This  solidifies  at 
once  and  seals  the  block  to  the  cover-slip. 

9.  Prepare  a  sterile  hanging-drop  slide,  and  smear 
hard  vaseline  or  melted  white  wax  on  the  rim  of  the 
metal  cell. 

10.  Invert  the  cover-slip  with  the  block  attached  on 
to  the  hanging-drop  slide,  and  seal  the  cover-slip  firmly 
in  place. 

11.  Observe  as  for  hanging-drop  cultivations. 


236  METHODS    OF   CULTIVATION 

ANAEROBIC  CULTIVATIONS. 

Numerous  methods  have  been  devised  for  the  culti- 
vation of  anaerobic  bacteria,  the  majority  requiring 
the  employment  of  special  apparatus.  The  principle 
upon  which  any  method  is  based  and  upon  which  it 
depends  for  its  success  falls  under  one  or  another  of  the 
following  headings : 

(a)  Exclusion  of  air  from  the  cultivation. 

(b)  Exhaustion  of  air  from  the  vessel  containing  the 
cultivation  by  means  of  an  air  pump — i.  e.,  cultivation 
in  vacuo. 

(c)  Absorption  of  oxygen  from  the  air  in  contact  with 
the  cultivation  by  means  of  pyrogallic  acid  rendered  alka- 
line with  caustic  soda — i.  e.,  cultivation  in  an  atmos- 
phere of  nitrogen. 

(d)  Displacement  of  air  by  an  indifferent  gas,  such 
as  hydrogen  or  coal  gas — i.  e.,  cultivation  in  an  atmos- 
phere of  hydrogen. 

(e)  A  combination  of  two  or  more  of  the  above 
methods. 

A  selection  of  the  simplest  and  most  generally  useful 
methods  is  given  here. 

Whenever  possible,  the  nutrient  media  that  are  em- 
ployed in  any  of  the  processes  should  contain  some 
easily  oxidisable  substance,  such  as  sodium  formate 
(0.4  per  cent.)  or  sodium  sulphindigotate  (o.i  per 
cent.),  which  will  absorb  all  the  available  oxygen  held 
in  solution  by  the  medium.  The  further  addition  of 
glucose,  2  per  cent.,  favors  the  growth  of  anaerobic 
bacteria  (vide,  pages  189-190). 

Further,  it  is  advisable  to  seal  all  joints  between 
india-rubber  stoppers  and  tubulures  or  the  mouths 
of  the  tubes  with  melted  paraffin;  glass  stoppers  and 
taps  should  be  lubricated  with  resin  ointment  or  a 
mixture  of  beeswax  i  part,  olive  oil  4  parts. 


ANAEROBIC     CULTURES  237 

(A)  Method  I  (Hesse's  Method)  .- 

1.  Make  a  stab  culture  in  gelatine  or  agar,  choosing 
for  the  purpose  a  straight  tube  containing  a  deep 
column    of   medium,    and   thrusting   the   inoculating 
needle  to  the  bottom  of  the  tube. 

2.  Pour  a  layer  of  sterilised  oil  (olive  oil,  vaseline,  or 
petroleum),  i  or  2  cm.  deep,  upon  the  surface  of  the 
medium. 

3.  Incubate. 

Method  II. — This   method   is   only   available   when 
dealing  with  pure  cultivations. 

1.  Liquefy  a  tube  of  gelatine   (or  agar)    by  heat, 
pour  it  into  a  Petri  dish,  and  allow  it  to  solidify. 

2.  Inoculate  the  surface  of  the  medium  in  one  spot 
only. 

3.  Remove  a  cover-slip  from  the  pot  of  absolute 
alcohol  with  sterile  forceps ;  burn  off  the  alcohol  in  the 
gas  flame. 

4.  Lower  the  now  sterile  cover-slip  carefully  on  to 
the  inoculated  surface  of  the  medium,  carefully  exclud- 
ing air  bubbles,  and  press  it  down  firmly  with  the 
points  of  the  forceps.     (A  sterile  disc  of  mica  may  be 
substituted  for  the  cover-slip.) 

5.  Incubate. 

Method  III  (Roux's  Physical  Method).— 

1.  Prepare  tube  cultures  of  fluid  media    (or  solid 
media  rendered  fluid  by  heat)  in  the  usual  way. 

2.  Aspirate  some  of  the  inoculated  media  into  capil- 
lary pipettes. 

3.  Seal  both  ends  of  each  pipette  in  the  blowpipe 
flame. 

4.  Incubate. 

Method  IV  (Roux's  Biological  Method).— 

1.  Plant  a  deep  stab,  as  in  method  I. 

2.  Pour  a  layer,  i  or  2  cm.  deep,  of  broth  cultivation 
of  a  vigourous  aerobe — e.  g.,  B.  aquatilis  sulcatus  or  B. 


238 


METHODS    OF   CULTIVATION 


prodigiosus  —  upon  the  surface  of  the  medium;  or  an 
equal  depth  of  liquefied  gelatine,  which  is  then  inocu- 
lated with  the  aerobic  organism. 

3.  Incubate. 

The  growth  of  the  aerobe  will  use  up  all  the  oxygen 
that  reaches  it  and  will  not  allow  any  to  pass  through 
to  the  medium  below,  which  will  consequently  remain 
in  an  anaerobic  condition. 

(B)  Method  V.- 

i  .  Prepare  tube  or  flask  cultivations  in  the  usual  way. 

2.  Replace  the  cotton-wool  plug  by 
an  india-rubber  stopper  perforated  with 
one  hole  and  fitted  with  a  length  of 
glass  tubing  which  has  a  constriction 
about  3  cm.  above  the  stopper  and  is 
then  bent  at  right  angles   (Fig.  129). 
The    stopper    and    glass    tubing    are 
sterilised  by  being  boiled  in  a  beaker 
of  water  for  five  minutes. 

3.  Connect   the    tube  leading  from 
the  culture  vessel  with  a  water  or  air 
pump,    interposing    a    WulfFs    bottle 
fitted  as  a  wash-bottle  and  containing 
sulphuric  acid. 

4.  Exhaust  the  air  from  the  culture 
vessel. 

5-  Before    disconnecting  the  appa- 
ratus, seal  the  glass  tube  from  the  cul- 
at  the    constriction,  using  the  blowpipe 


FlG' 


ture  vessel 
flame. 

6.  Incubate. 

(C)  Method  VI  (Buchner's  Method)  .- 

Apparatus  and  Solutions  Required.  — 

Buchner's  tube  (a  stout  glass  test-tube  23  cm.  long  and  4  cm. 
in  diameter,  fitted  with  india-rubber  stopper,  Fig.  130). 

Pyrogallic  acid  in  compressed  tablets  each  containing  i  gram. 
Dekanormal  solution  of  caustic  soda. 


ANAEROBIC    CULTURES 


239 


METHOD.  — 

1.  Prepare  the  tube  cultivation  in  the  usual  way. 

2.  Moisten  the  india-rubber  stopper  of  the  Buchner's 
tube  with  water  and  see  that  it  fits  the  mouth  of  the 
tube  accurately. 

3  .  Remove  the  stopper  from  the  caustic  soda  bottle. 

4.  Drop  one  of  the  pyrogallic  acid  tablets  into  the 
Buchner  's  tube  (roughly,  use  i  gramme  pyrogallic  acid 
for  every  100  c.c.  air  capacity  of  the  receiving  vessel). 

5.  Add  about  i  c.c.  of  the  soda  solution. 

6.  Place  the  inoculated  tube  inside  the  Buchner's 
tube.     The  pyrogallic  tablet  acts  as  a 

buffer  and  prevents  damage  to  either 
the  inoculated  tube  or  the  Buchner's 
tube  even  should  it  be  slipped  in  hur- 
riedly. 

7.  Fit  the  india-rubber  stopper  tightly 
into  the  mouth  of  the  Buchner  's  tube. 

The  pyrogallic  acid  tablet  dissolves 
slowly  in  the  soda  solution  and  its  oxida- 
tion proceeds  very  slowly  at  first  so  that 
ample  time  is  available  when  this  method 
is  adopted. 

8.  Restopper  the  caustic  soda  bottle. 
'9.  Place  Buchner's  tube  in  a  wire  sup- 

port, and  incubate. 

Method  VII  (Wright  's  Method)  .— 

1.  Prepare    tube    cultivation    in    the 
usual  way. 

2.  Cut  off  that  portion  of  the  cotton-wool  plug  project- 
ing above  the  mouth  of  the  tube  with  scissors,  then 
push  the  plug  into  the  tube  for  a  distance  of  2  or  3  cm. 

1  If  compressed  tablets  of  pyrogallic  acid  cannot  be  obtained  make  up  a 
stock  "acid  pyro"  solution 

Pyrogallic  acid,  10  grammes 

Hydrochloric  acid,  1.5  c.c. 

Distilled  water,  100  c.c. 
and  at  step  4,  run  in  10  c.c.  of  the  solution. 


240  METHODS    OF   CULTIVATION 

3 .  By  means  of  a  pipette  drop  about  i  c.c.  of  pyrogal- 
lic  acid  10  per  cent,  aqueous  solution  on  to  the  plug. 
It  will  immediately  be  absorbed  by  the  cotton- wool. 

4.  With  another  pipette  run  in  an  equal  quantity 
of  the  caustic  soda  solution. 

5.  Quickly  close  the  mouth  of  the  tube  with  a  tightly 
fitting  india-rubber  stopper. 

6.  Incubate. 

Method  VIII  (McLeod's  Method).— 

Apparatus  and  Solutions  Required. — 

McLeod's  plate  base  (a  hollow  glazed  earthenware  disc  9  cm.  in 
diameter  and  2  cm.  deep :  the  upper  surface  is  pierced  by  a  central 
hole,  2  cm.  in  diameter,  giving  access  to  the  interior,  the  lower 
part  of  which  is  divided  into  two  by  a  low  partition.  A  shallow 
groove  encircles  the  upper  surface  near  to  the  edge) . 


FlG.  131. — McLeod's   anaerobic   plate  base  with  half  petri  dish  inverted 

in  situ 

Plasticine. 

Pyrogallic  acid  (i  gramme)  compressed  tablets. 
Sodic  hydroxide  (0.4  gramme)  compressed  tablets. 
Wash  bottle  of  distilled  water. 

Surface  plates  of  one  or  other  agar  medium  (in  petri  dishes  of 
8  cm.  diameter). 
Surface  plate  spreader. 

METHOD.— 

i.  Roll  out  a  long  cylinder  of  plasticine  and  fit  it 
into  the  groove  on  the  upper  surface  of  the  earthenware 
base. 


ANAEROBIC     CULTURES  241 

2.  Place  a  tablet  of  pyrogallic  acid  in  one  division 
of  the  interior  of  the  plate  base,  and  two  tablets  of 
sodic  hydroxide  in  the  other. 

3.  Prepare  surface  plate  culture  of  the  organism  to 
be  cultivated. 

4.  Run  a  few  cubic  centimetres  of  distilled  water 
into  that   division  of  the  plate  base  containing  the 
sodic  hydroxide. 

5.  Invert  the  bottom  half  of  the  surface  plate  over 
the  plate  base  and  press  its  edges  firmly  down  into  the 
plasticine  filling  the  groove. 

6.  Label  and  incubate. 

(D)  Method  IX.- 

Apparatus  Required. — 

Small  Ruffer's  or  Woodhead's  flask  (Fig.  33). 

Sterile  india-rubber  stopper. 

India-rubber  tubing. 

Glass  tubing. 

Metal  screw  clips. 

Cylinder  of  compressed  hydrogen ;  or  hydrogen  gas  apparatus 

METHOD.— 

1 .  Sterilise  a  glass  vessel,  shaped  as  in  a  Ruffer  's  or 
Woodhead's  flask,  in  the  hot-air  oven.     (The  tubulure 
and  the  side  tubes  are  plugged  with  cotton-wool.) 
After  sterilisation,  fix  a  short  piece  of  rubber  tubing 
occluded  by  a  metal  clip  to  each  side  tube. 

2.  Inoculate  a  large  quantity  (e.  g.,  200  c.c.)  of  the 
medium.     Where  solid  media  are  employed  they  must 
first  be  liquefied  by  heat. 

3.  Remove  the  cotton-wool  plug  from  the  tubulure 
and  pour  the  inoculated  medium  into  the  glass  vessel. 

4.  Close  the  tubulure  by  means  of  an  india-rubber 
stopper  previously  sterilised  by  boiling  in  a  beaker  of 
water. 

5.  Connect  up  the  india-rubber  tubing  on  one  of 
the  side  tubes  with  a  cylinder  of  compressed  hydrogen 

16 


242 


METHODS   OF  CULTIVATION 


a  be 

FIG.  132. — Kipp's  hydrogen  apparatus,  (a)  connected  up  to  two  washing 
bottles  containing  (I)  lead  acetate  10  per  cent,  solution,  to  remove  H2S 
and  (c)  silver  nitrate  solution  to  remove  AsH3.  A  third  washing  bottle 
containing  pyrogallic  acid  10  per  cent,  solution,  rendered  alkaline,  to 
remove  any  trace  of  oxygen,  is  sometimes  introduced. 


FIG.  133. — Improved  gas  apparatus;  the  metal  is  contained  in  a  perforated 
glass  tube  which  is  submerged  in  acid  when  the  triangular  bottle  is  upright 
(a),  but  is  above  the  level  of  the  liquid  when  the  bottle  is  turned  on  its 
side  (6). 


ANACROBIC     CULTURES  243 

(or  the  delivery  tube  of  a  Kipp's  Fig.  132  or  other 
hydrogen  apparatus,  Fig.  133),  interposing  a  short 
piece  of  glass  tubing;  and  in  like  manner  connect  a 
long  piece  of  rubber  tubing  which  should  be  led  into 
a  basin  of  water,  to  the  opposite  side  tube. 

6.  Open  both  metal  clips  and  pass  hydrogen  through 
the  vessel  until  the  atmospheric  air  is  replaced  by 
hydrogen.     This  is  determined  by  collecting  some  of 
the  gas  which  bubbles  through  the  water  in  the  basin 
in  a  test-tube  and  testing  it  by  means  of  a  lighted  taper. 

7.  Close  the  metal  clip  on  the  tube  through  which 
the  gas  is  entering ;  close  the  clip  on  the  exit  tube. 

8.  Disconnect  the  gas  apparatus. 

9.  Incubate. 

Method  X  (Botkin's  Method) .- 

Apparatus  Required. — 

Large  glass  dish  20  cm.  diameter  and  8  cm.  deep.     Flat  leaden 
cross  slightly  shorter  than  the  internal  diameter  of  the  glass  dish. 
Bell  glass  about  15  cm.  diameter  and  20  to  25  cm.  high. 
Metal  frame  for  plate  cultivations. 
Or,  glass  battery  jar  for  tube  cultivations. 
Cylinder  of  compressed  hydrogen. 
Rubber  tubing. 

Two  pieces  of  U-shaped  glass  tubing  (each  arm  8  cm.  in  length). 
Half  a  litre  of  glycerine  (or  metallic  mercury). 

METHOD.— 

1.  Place  the  leaden  cross  inside  the  glass  dish,  resting 
on  the  bottom. 

2.  Prepare  the  cultivations  in  the  usual  way. 

3.  Place  the  tube  cultivations  in  a  glass  battery  jar 
(or  the  plate  cultivations  on  a  metal  frame) ,  resting  on 
the  centre  of  the  leaden  cross. 

4.  Cover  the  cultivations  with  the  bell  jar. 

5.  Adjust  the  U-shaped  pieces  of  glass  tubing  in  a 
vertical  position  on  opposite  sides  of  the  bell  jar,  one 
arm  of  each  inside  the  jar,  the  other  outside.     These 
tubes  are  best  held  in  position  by  embedding  the  U- 


244 


METHODS    OF   CULTIVATION 


shaped  bends  in  two  lumps  of  plasterine  stuck  on  the 
bottom  of  the  glass  dish.  Fix  a  short  length  of  rubber 
tubing  clamped  with  a  metal  clip  to  each  of  the  out- 
side arms  (Fig.  134). 

6.  Fill   the   glass   dish   with  glycerine   or  metallic 
mercury  to  a  depth  of  about  5  cm. 


FIG.  134. — Botkin's  apparatus. 

7.  Connect  up  one  U-shaped  tube  with  the  hydrogen 
cylinder  (or  gas  apparatus)  by  means  of  rubber  tubing. 
Replace  the  atmospheric  air  by  hydrogen,  as  in  method 
IX. 

8.  Clamp  the  tubes  and  disconnect  the  gas  apparatus. 

9.  Incubate. 

Method  XI  (Novy's  Method).— 
Apparatus  Required. — 

Jar  for  plate  cultivations  (Fig.  135). 
Or,  jar  for  tube  cultivations  (Fig.  136). 
Lubricant  for  stopper  of  jar. 
Rubber  tubing. 
Cylinder  of  compressed  hydrogen. 


ANAEROBIC    CULTURES 


245 


METHOD. — 

1.  Prepare  cultivations  in  the  usual  way. 

2.  Place  these  inside  the  jar. 

3.  Lubricate  the  stopper  and  insert  it  in  the  mouth 
of  the  jar,  with  the  handle  in  a  line  with  the  two  side 
tubes. 

4.  Connect  up  the  delivery  tube  a  with  the  hydrogen 
gas  supply  by  means  of  rubber  tubing. 


FIG.  135. — Novy's  jar  for 
plate  cultivations. 


FIG.  136. — Novy's  jar  for 
tube  cultivations. 


5.  Attach  a  piece  of  rubber  tubing  to  the  exit  tube 
b  and  collect  samples  of  the  issuing  gas  (over  water) 
and  test  from  time  to  time. 

6.  When  the  air  is  completely  displaced  by  hydrogen, 
turn  the  handle  of  the  stopper  at  right  angles  to  the 
line  of  entry  and  exit  tubes;  this  seals  the  orifice  of 
both  tubes. 

7.  Disconnect  the  gas  apparatus  and  incubate. 

(E)  Method  XII  (Bulloch's  Method).— 

Apparatus  Required. — 

Bulloch's  jar. 

Pot  of  resin  ointment. 

Small  glass  dish  14  cm.  diameter  by  5  cm.  deep. 

Vessel  for  tube  cultures  or  metal  rack  for  plate  cultures. 


246  METHODS    OF   CULTIVATION 

Pyrogallic  acid  tablets. 
Cylinder  of  compressed  hydrogen. 
.  Geryk  or  other  air  pump. 
Rubber  pressure  tubing. 
10  c.c.  pipette. 
Glass  tubing. 

Dry  granulated  caustic  soda  or  compressed  tablets  each  con- 
taining 0.4  grammes  sodic  hydroxide. 
Small  beaker  of  water. 

METHOD.— 

1.  Prepare  the  cultivations  in  the  usual  way. 

2.  Place  the  glass  dish  in  the  centre  of  the  glass 
slab,  and  stand  the  cultivations  inside  this. 

3.  Place    a    sufficient    number    of    pyrogallic    acid 
tablets  at  one  side  of  the  glass  dish  (i.  e.,  i  tablet  for 
each  100  cubic  centimeters  air  capacity  of  the  bell  jar). 
Place  a  small  heap  of  dry  granulated  soda  (or  half  a 
dozen  tablets  of  sodic  hydroxide)  by  the  side  of  the 
pyro  tablets. 

4.  Smear  the  flange  of  the  bell  jar  with  resin  oint- 
ment and  apply  the  jar  firmly  to  the  glass  slab,  covering 
the  cultivations — so  arranged  that  the  long  tube  passes 
with  its  lower  end  into  the  glass  dish  at  a  point  directly 
opposite  to  the  pyrogallic  acid  tablets.     Lubricate  the 
two  stop-cocks  with  resin  ointment  (Fig.  137). 

5.  Connect  up  the  short  tube  a  with  the  gas-supply 
by  means  of  rubber  pressure  tubing  and  open  both  stop- 
cocks. 

6.  Connect  a  long,  straight  piece  of  glass  tubing  to 
the  long  tube  b  by  means  of  a  piece  of  rubber  tubing 
interposing  a  screw  clamp :  and  collect  samples  of  the 
issuing  gas  from  time  to  time  and  test. 

7.  When  the  air  is  displaced,  shut  off  the  stop-cock 
of  the  entry  tube,  then  that  of  the  exit  tube  b.     Screw 
down  the  clamp  and  remove  the  glass  tube  from  the 
rubber  connection  and  connect  up  the  short  tube  a  to 
the  air  pump  by  means  of  pressure  tubing. 

8.  Open  the  stop-cock  of  tube  a  and  with  two  or  three 


ANAEROBIC    CULTURES 


247 


strokes  of  the  air  pump,  aspirate  a  small  quanity  of 
gas,  so  creating  a  slight  vacuum.  Then  shut  off  the 
stop-cock  and  disconnect  the  air  pump. 

9.  Fill  the  10  c.c.  bulb  pipette  with  water;  insert  its 
point  into  the  rubber  tubing  on  the  long  tube  b  as  far  as 
the  screw  clamp.  Open  the  screw  clamp  and  run  in 
water  until  stopped  by  the  internal  pressure.  Shut 
off  stop-cock.  (The  water  dissolves  the  soda  and 


FIG.  137. — Bulloch's  jar. 

pyrogallic  acid  converting  the  latter  into  alkaline 
pyro.  and  so  bringing  its  latent  capacity  for  oxygen 
into  action) . 

10.  Reverse  the  tubes  from  the  tubulures  so  that 
they  meet,  out  of  harm's  way,  over  the  top  of  the 
bell  glass ;  again  see  that  all  joints  are  tight  and  trans- 
fer the  apparatus  to  the  incubator. 

This  last  method  is  the  most  satisfactory  for  anae- 
robic cultivations,  as  by  its  means  complete  anaerobi- 
osis  can  be  obtained  with  the  least  expenditure  of  time 
and  trouble. 


XV.  METHODS  OF  ISOLATION. 

THE  work  in  the  preceding  sections,  arranged  to 
demonstrate  the  chief  biological  characters  of  bacteria 
in  general,  is  intended  to  be  carried  out  by  means  of 
cultivations  of  various  organisms  previously  isolated 
and  identified  and  supplied  to  the  student  in  a  state  of 
purity.  A  cultivation  which  comprises  the  prog- 
eny of  a  single  cell  is  termed  a  "pure  culture";  one 
which  contains  representatives  of  two  or  more  species 


FIG.  138. — Haematocytometer  cell,  showing,  a,  section  through  the  centre  of 
the  cell,  and  &,  a  magnified  image  of  the  cell  rulings. 

of  bacteria  is  spoken  of  as  an  "impure, "  or  "mixed" 
"cultivation,"  and  it  now  becomes  necessary  to  indi- 
cate the  chief  methods  by  which  one  or  more  organisms 
may  be  isolated  in  a  state  of  purity  from  a  mixture; 
whether  that  mixture  exists  as  an  impure  laboratory 
cultivation,  or  is  contained  in  pus  and  other  morbid 
exudations,  infected  tissues,  or  water  or  food-stuffs. 

Before  the  introduction  of  solid  media  the  only 
method  of  obtaining  pure  cultivations  was  by  "dilu- 
tion"— by  no  means  a  reliable  method.  "Dilution" 
consisted  in  estimating  approximately  the  number  of 
bacteria  present  in  a  given  volume  of  fluid  (by  means 
of  a  graduated-celled  slide  resembling  a  haemato- 

248 


BIOLOGICAL    DIFFERENTIATION  249 

cytometer,  Fig.  138),  and  diluting  the  fluid  by  the  addi- 
tion of  sterile  water  or  bouillon  until  a  given  volume 
(usually  i  c.c.)  of  the  dilution  contained  but  one 
organism.  By  planting  this  volume  of  the  fluid  into 
several  tubes  or  flasks  of  nutrient  media,  it  occasion- 
ally happened  that  the  resulting  growth  was  the  pro- 
duct of  one  individual  microbe.  A  method  so  uncer- 
tain is  now  fortunately  replaced  by  many  others, 
more  reliable  and  convenient,  and  in  those  methods 
selected  for  description  here,  the  segregation  and  isola- 
tion of  the  required  bacteria  may  be  effected — 

A.  By  Mechanical  Separation. 

i.  By  surface  plate  cultivation: 

(a)  Gelatine. 

(b)  Agar. 

(c)  Serum  agar. 

(d)  Blood  agar. 

(e)  Hanging-drop  or  block. 
[2.  By  Esmarch's  roll  cultivation: 

This  archaic  method  (see  page  226)  is  no  longer 
employed  for  the  isolation  of  bacteria.] 

3.  By  serial  cultivation. 

B.  By  Biological  Differentiation. 

4.  By  differential  media. 

(a)  Selective. 

(b)  Deterrent. 

5.  By  differential  incubation. 

6.  By  differential  sterilisation. 

7.  By  differential  atmosphere  cultivation. 

8.  By  animal  inoculation. 

The  selection  of  the  method  to  be  employed  in  any 
specific  instance  will  depend  upon  a  variety  of  cir- 
cumstances, and  often  a  combination  of  two  or  more 
will  ensure  a  quicker  and  more  reliable  result  than  a 
rigid  adherence  to  any  one  method.  Experience  is 
the  only  reliable  guide,  but  as  a  general  rule  the  use  of 


250 


METHODS    OF   ISOLATION 


either  the  first  or  the  third  method  will  be  found  most 
convenient,  affording  as  each  of  them  does  an  opportun- 
ity for  the  simultaneous  isolation  of  several  or  all 
of  the  varieties  of  bacteria  present  in  a  mixture. 

1.  Surface  Plate  Cultivations. — 

(a)  Gelatine  (vide  page  164). 

(b)  Agar  (vide  page  167).  • 

(c)  Alkaline  serum  agar  (vide  page  211). 

These  plates  are  prepared  in  a  manner  precisely 
similar  to  that  adopted  for  nutrient  gelatine  and 
agar  surface  plates  (vide  pages  231-233). 

(d)  Serum  Agar. — 

1.  Melt  three  tubes  of  nutrient  agar,  label  them  1,2, 
and  3,  and  place  them,  with  three  tubes  of  sterile 
fluid  serum,  also  labelled  ia,  20,  and  30,  in  a  water-bath 
regulated  at  45°  C. ;  allow  sufficient  time  to  elapse  for 
the  temperature  of  the  contents  of  each  tube  to  reach 
that  of  the  water-bath. 

2.  Take  serum  tube  No.  la  and  agar  tube  No.   i. 
Flame  the  plugs  and  remove  them  from  the  tubes 
(retaining  the  plug  of  the  agar  tube  in  the  hand) ; 
flame  the  mouths  of  the  tubes,  pour  the  serum  into  the 
tube  of  liquefied  agar  and  replace  the  plug  of  the  agar 
tube. 

3.  Mix  thoroughly  and  pour  plate  No.  i  secundum 
artem. 

4.  Treat  the  remaining  tube  of  agar  and  serum  in  a 
similar  fashion,  and  pour  plates  Nos.  2  and  3. 

5.  Dry  the  serum  agar  plates  in  the  incubator  running 
at  60°  C.  for  one  hour  (see  page  232). 

6.  Inoculate  the  plates  in  series  as  described  for 
gelatine  surface  plates   (page  231). 

(e)  Blood  Agar,  Human.— 

i.  Melt  a  tube  of  sterile  agar  and  pour  it  into  a  sterile 
plate;  let  it  set. 


SERIAL    CULTIVATIONS  251 

2.  Collect  a  few  drops  of  human  blood,  under  all 
aseptic  conditions,  in  a  sterile  capillary  teat  pipette. 

3.  Raise  the  cover  of  the  Petri  dish  very  slightly, 
insert  the  extremity  of  the  capillary  pipette,  and  deposit 
the  blood  on  the  centre  of  the  agar  surface.     Close  the 
dish. 

4.  Charge  a  platinum  loop  with  a  small  quantity  of 
the  inoculum.     Raise  the  cover  of  the  plate,  introduce 
the  loop,   mix  its  contents   with  the  drop  of  blood, 
remove  the  loop,  close  the  dish  and  sterilise  the  loop. 

5.  Finally  smear  the  mixture  over  the  surface  of  the 
agar  with  a  sterilised  L-shaped  rod. 

6.  Label  and  incubate. 

(If  considered  necessary,  two,  three,  or  more  similar 
plates  may  be  inoculated  in  series.) 

(/)  Blood  Agar,  Animal. — 

When  preparing  titrated  blood  agar  (page  171)  it  is 
always  advisable  to  pour  several  blood  agar  tubes  into 
plates,  which  can  be  stored  in  the  ice  chest  ready  for 
use  at  any  moment  for  surface  plate  cultures. 

(g)  Hanging-drop  or  block  culture,  (vide  page  233). 

3.  Serial  Cultivations. — These  are  usually  made  upon 
agar  or  blood-serum,  although  gelatine  may  also  be  used. 
The  method  is  as  follows : 

1.  Take  at  least  four  "slanted"  tubes  of  media  and 
number  them  consecutively. 

2.  Flame  all  the  plugs  and  see  that  each  can  be 
readily  removed. 

3.  Charge  the  platinum  loop  with  a  small  quantity 
of  the  inoculum,  observing  the  usual  routine,  and  plant 
tube  No.  i,  smearing  thoroughly  all  over  the  surface. 
If   any   water   of   condensation   has  collected  at  the 
bottom   of    the  tube,   use    this    as  a  diluent   before 
smearing  the  contents  of  the  loop  over  the  surface  of 
the  medium. 

4.  Without  sterilising  or  recharging  the  loop,  inocu- 


252 


METHODS    OF   ISOLATION 


late  tube  No.  2,  by  making  three  parallel  streaks  from 
end  to  end  of  the  slanted  surface. 

5.  Plant  the  remainder  of  the  tubes  in  the  series  as 
"smears"  like  tube  No.  i. 

6.  Label  with  distinctive  name  or  number,  and  date; 
incubate. 

The  growth  that  ensues  in  the  first  two  or  three 
tubes  of  the  series  will  probably  be  so  crowded  as  to  be 
useless.  Toward  the  end  of  the  series,  however,  dis- 
crete colonies  will  be  found,  each  of  which  can  be 
transferred  to  a  fresh  tube  of  nutrient  medium  without 
risk  of  contamination  from  the  neighbouring  colonies. 

"Working"  up  Plates.— 

Having  succeeded  in  obtaining  a  plate  (or  tube 
cultivation)  in  which  the  colonies  are  well  grown  and 
sufficiently  separated  from  each  other,  the  process  of 
"  working  up,"  "  pricking  out,"  or  "  fishing  "  the  colonies 
in  order  to  obtain  subcultures  in  a  state  of  purity  from 
each  of  the  different  bacteria  present  must  now  be 
proceeded  with. 

Occasionally  it  happens  that  this  is  quite  a  simple 
matter.  For  example,  the  original  mixed  cultivation 
when  examined  microscopically  was  found  to  contain  a 
Gram  positive  micrococcus,  a  Gram  positive  straight 
bacillus  and  a  Gram  negative  short  bacillus.  The  third 
gelatine  plate  prepared  from  this  mixture,  on  inspection 
after  four  day's  incubation,  showed  twenty-five  colonies 
— seven  moist  yellow  colonies,  each  sinking  into  a  shal- 
low pit  of  liquefied  gelatine,  fourteen  flat  irridescent 
filmy  colonies,  and  four  raised  white  slimy  colonies. 
A  film  preparation  (stained  Gram)  from  each  variety  ex- 
amined microscopically  showed  that  the  yellow  liquefy- 
ing colony  was  composed  of  Gram  positive  micrococci ; 
the  flat  colony  of  Gram  positive  bacilli  and  the  white 
colony  of  gram  negative  bacilli.  One  of  each  of  these 
varieties  of  colonies  would  be  transferred  by  means  of 


FISHING    COLONIES  253 

the  sterilised  loop  to  a  fresh  gelatine  culture  tube,  and 
after  incubation  the  growth  in  each  subculture  would 
correspond  culturally  and  microscopically  with  that  of 
the  plate  colony  from  which  it  was  derived, — the  object 
aimed  at  would  therefore  be  achieved. 

Usually,  however,  the  colonies  cannot  be  thus  readily 
differentiated,  and  unless  they  are  "worked  up"  in  an 
orderly  and  systematic  manner  much  labour  will  be 
vainly  expended  and  valuable  time  wasted.  The  fol- 
lowing method  minimises  the  difficulties  involved. 

(A)  Inspection. 

a.  Without  opening  the  plate  carefully  study  the 
various  colonies  with  the  naked  eye,  with  the  assistance 
of  a  watchmaker's  lens  or  by  inverting  the  plate  on 
the  stage  of   the   microscope   and   viewing   with   the 
i -inch  objective  through  the  bottom  of  the  plate  and 
the  layer  of  medium. 

b.  If  gross  differences  can  be  detected  mark  a  small 
circle  on  the  bottom  of  the  plate  around  the  site  of 
each  of  the  selected  colonies,  with  the  grease  pencil. 

c.  If  no  obvious  differences  can  be  made  out  choose 
nine  colonies  haphazard  and  indicate  their  positions  by 
pencil  marks  on  the  bottom  of  the  plate. 

(B)  Fishing  Colonies.— 

a.  Take  a  sterile  Petri  dish  and  invert  it  upon  the 
laboratory    bench.     Rule   two    parallel    lines    on    the 
bottom  of  the  dish  with  a  grease  pencil,  and  two  more 
parallel  lines  at  right  angles  to  the  first  pair — so  dividing 
the  area  of  the  dish  into  nine  portions.     Number  the 
top  right-hand  portion  i,  and  the  central  bottom  por- 
tion 8  (Fig.  139).     Revert  the  dish.     The  numbers  i 
and  8  can  be  readily  recognised  through  the  glass  and 
by  their  positions  enable  any  of  the  other  divisions  to 
be  localised  by  number.     This  is  the  stock  dish. 

b.  Slightly  raise  the  cover  of  the  dish,  and  with  a 


254 


METHODS    OF   ISOLATION 


sterile  teat-pipette  deposit  a  small  drop  of  sterile  water 
in  the  centre  of  each  of  the  nine  divisions. 

c.  With  the  sterilised  platinum  spatula  raise  one  of 
the  marked  colonies  from  the  "  plate  3  "  and  transfer  it 
to  the  first  division  in  the  ruled  plate  and  emulsify  it 
in  the  drop  of  water  awaiting  it.  Repeat  this  process 
with  the  remaining  colonies,  emulsifying  a  separate 
colony  in  each  drop  of  water. 

(C)  Preliminary  Differentiation  of  Bacteria. — 

a.  Prepare  a  cover-slip  film  preparation  from  each 
drop  of  emulsion  in  the  " stock  dish"  and  number  to 

correspond  to  the  division 
from  which  it  was  taken. 
Stain  by  Gram's  method. 

b.     Examine        microscop- 
ically, using  the  oil  immersion 
lens  and  note  the  numbers  of 
those   cover-slips  which  mor- 
phologically and  by  Gram  re- 
sults appear  to  be  composed 
FIG.  i39.-Diagram  for  stock    of    different   species    of    bac- 
Plate-  teria. 

(D)  Preparing  Isolation  Subcultures. — 

a.  Inoculate  an  agar  slope  and  a  broth  tube  from  the 
emulsion  in  the  stock  dish  corresponding  to  each  of 
these  specially  selected  numbers. 

b.  Ascertain  whether  the  cover-slips  from  the  nine 
emulsions   in  the   stock  dish  include  all  the  varieties 
represented  in  the  cover-slip  film  preparation  made  from 
the  original  mixture  before  plating. 

c.  If  some  varieties  are  missing  prepare  a  second 
stock  dish  from  other  colonies  on  plate  3,  and  repeat 
the  process  until  each  morphological  form  or  tinctorial 
variety  has  been  secured  in  subculture. 

d.  Place  the  stock  dishes  in  the  ice  chest  to  await  the 


8 


DIFFERENTIAL    INCUBATION  255 

results  of  incubation.  (If  any  of  the  subcultures  fail, 
further  material  can  be  obtained  from  the  correspond- 
ing emulsion ;  or  if  it  has  dried,  by  moistening  it  with  a 
further  drop  of  sterile  distilled  water.) 

e.  Incubate   all   the   subcultures   and  identify  the 
organisms  picked  out. 

4.  Differential  Media.— 

(a)  Selective. — Some  varieties  of  media  are  specially 
suitable  for  certain  species  of  bacteria  and  enable  them 
to  overgrow  and  finally  choke  out  other  varieties;  e.  g., 
wort  is  the  most  suitable  medium-base  for  the  growth 
of  torulae  and  yeasts  and  should  be  employed  when 
pouring  plates  for  the  isolation  of  these  organisms.     To 
obtain  a  pure  cultivation  of  yeast  from  a  mixture  con- 
taining bacteria  as  well,  it  is  often  sufficient  to  inoculate 
wort  from  the  mixture  and  incubate  at  37°  C.   for 
twenty-four  hours.     Plant  a  fresh  tube  of  wort  from 
the  resulting  growth  and  incubate.     Repeat  the  proc- 
ess once  more,  and  from  the  growth  in  this  third  tube 
plant  a  streak  on  wort  gelatine,  and  incubate  at  20°  C. 
The  resulting  growth  will  almost  certainly  be  a  pure 
culture  of  the  yeast. 

(b)  Deterrent. — The    converse    of    the    above  also 
obtains.     Certain  media  possess  the  power  of  inhibiting 
the  growth  of  a  greater  or  less  number  of  species. 
For  instance,  media  containing  carbolic  acid  to  the 
amount  of  i  per  cent,  will  inhibit  the  growth  of  prac- 
tically everything  but  the   Bacillus    coli    communis. 

5.  Differential  Incubation. — In  isolating  certain  bac- 
teria,  advantage  is  taken  of  the  fact  that  different 
species  vary  in  their  optimum  temperature.     A  mix- 
ture containing  the  Bacillus  typhosus  and  the  Bacillus 
aquatilis  sulcatus,   for  example,   may  be  planted  on 
two  slanted  agar  tubes,  the  one  incubated  at  40°  C., 
and  the  other  at    12°  C.     After  twenty-four  hours' 
incubation  the  first  will  show  a  pure  cultivation  of  the 


256  METHODS    OF   ISOLATION 

Bacillus  typhosus,  whilst  the  second  will  be  an  almost 
pure  culture  of  the  Bacillus  aquatilis. 

6.  Differential  Sterilisation.— 

(a)  Non-sporing  Bacteria. — Similarly,  advantage  may 
be  taken  of  the  varying  thermal  death-points  of  bac- 
teria.    From  a  mixture  of  two  organisms  whose  ther- 
mal death-points  differ  by,  say,  4°  C. — e.  g.,  Bacillus 
pyocyaneus,  thermal  death-point  55°  C.,  and  Bacillus 
mesentericus  vulgatus,  thermal  death-point  60°  C. — a 
pure  cultivation  of  the  latter  may  be  obtained  by  heat- 
ing the  mixture  in  a  water-bath  to  58°  C.  and  keeping 
it  at  that  point  for  ten 'minutes.     The  mixture  is  then 
planted  on  to  fresh  media  and  incubated,  when  the  re- 
sulting growth  will  be  found  to  consist  entirely  of  the 
B.  mesentericus. 

(b)  Sparing  Bacteria. — This  method  finds  its  chief 
practical  application  in  the  differentiation   of  a  spore- 
bearing  organism  from  one  which  does  not  form  spores. 
In  this  case  the  mixture  is  heated  in  a  water-bath  at 
80°  C.  for  fifteen  to  twenty  minutes.     At  the  end  of 
this  time  the  non-sporing  bacteria  are  dead,  and  cul- 
tivations made  from  the  mixture  will  yield  a  growth 
resulting  from  the  germination  of  the  spores  only. 

Differential  sterilisation  at  80°  C.  is  most  conveni- 
ently carried  out  in  a  water-bath  of  special  construction, 
designed  by  Balfour  Stewart  (Fig.  140) .  It  consists  of  a 
double-walled  copper  vessel  mounted  on  legs,  and  pro- 
vided with  a  tubulure  communicating  with  the  space 
between  the  walls.  This  space  is  nearly  filled  with 
benzole  (boiling-point  80°  C. ;  pure  benzole,  free  from 
thiophene  must  be  employed  for  the  purpose,  otherwise 
the  boiling-point  gradually  and  perceptibly  rises  in  the 
course  of  time),  and  to  the  tubulure  is  fitted  a  long 
glass  tube,  some  2  metres  long  and  about  0.75  cm. 
diameter,  serving  as  a  condensing  tube  (a  tube  half 
this  length  if  provided  with  a  condensing  bulb  at 


DIFFERENTIAL   ATMOSPHERE    CULTIVATION 


257 


the  centre  will  be  equally  efficient).  The  interior 
of  the  vessel  is  partly  filled  with  water  and  covered  with 
a  lid  which  is  perforated  for  a  thermometer.  This 
latter  dips  into  the  water  and  records  its  temperature. 
A  very  small  Bunsen  flame  under  the  apparatus  suffices 
to  keep  the  benzole  boiling  and  the  water  within  at  a 
constant  temperature  of  80°  C.  The  bath  is  thus  always 
ready  for  use. 

METHOD. — To  use  the  apparatus. 

1 .  Place  some  of  the  mixture  itself, 
if  fluid,  containing  the  spores,  or  an 
emulsion    of    the    same    if    derived 
from  solid  material,  in  a  test-tube. 

2.  Immerse    the    test-tube  in  the 
water  contained  in  the  benzole  bath, 
taking   care  that  the  upper  level  of 
the  liquid  in  the  tube  is  at  least  2 
cm.  beneath  the  surface  of  the  water 
in  the  copper  vessel. 

3.  The  temperature  of  the  water, 
of  course,   falls  a  few  degrees  after 
opening  the  bath    and  introducing  a 
tube  of  colder  liquid,  but  after  a  few 
minutes  the   temperature   will  have 
again  reached  80°  C. 

4.  When   the    thermometer   again 
records    80°    C.,    note  the  time,  and 
fifteen  minutes  later  remove  the  tube 
containing  the  mixture  from  the  bath. 

5.  Make  cultures  upon  suitable  media;  incubate. 

7.  Differential  Atmosphere  Cultivation. — 

(a)  By  adapting  the  atmospheric  conditions  to  the 
particular  organism  it  is  desired  to  isolate,  it  is  com- 
paratively easy  to  separate  a  strict  aerobe  from  a 
strict  anaerobe,  and  vice  versa.  In  the  first  case, 
however,  it  is  important  that  the  cultivations  should 
17 


FIG.  140. — Benzole 
bath. 


258  METHODS    OF   ISOLATION 

be  made  upon  solid  media,  for  if  carried  out  in  fluid 
media  the  aerobes  multiplying  in  the  upper  layers  of 
fluid  render  the  depths  completely  anaerobic,  and 
under  these  conditions  the  growth  of  the  anaerobes  will 
continue  unchecked. 

(6)  When  it  is  desired  to  separate  a  facultative 
anaerobe  from  a  strict  anaerobe,  it  is  generally  suffi- 
cient to  plant  the  mixture  upon  the  sloped  surface 
agar,  incubate  aerobically  at  37°  C.,  and  examine 
carefully  at  frequent  intervals.  At  the  first  sign  of 
growth,  subcultivations  must  be  prepared  and  treated 
in  a  similar  manner.  As  a  result  of  these  rapid  subcul- 
tures, the  facultative  anaerobe  will  be  secured  in 
pure  culture  at  about  the  third  or  fourth  generation. 

(c)  If,  on  the  other  hand,  the  strict  anaerobe  is  the 
organism  required  from  a  mixture  of  facultative  and 
strict  anaerobes,  pour  plates  of  glucose  formate  agar 
(or  gelatine)  in  the  usual  manner,  place  them  in  a 
Bulloch's  or  Novy's  jar,  and  incubate  at  a  suitable 
temperature.  Pick  off  the  colonies  of  the  required 
organism  when  the  growth  appears,  and  transfer  to 
tubes  of  the  various  media. 

Incubate  under  suitable  conditions  as  to  temperature 
and  atmosphere. 

8.  Animal  Inoculation. — Finally,  when  dealing  with 
pathogenic  organisms,  it  is  often  advisable  to  inoculate 
some  of  the  impure  culture  (or  even  some  of  the  original 
materies  morbi)  into  an  animal  specially  chosen  on  ac- 
count of  its  susceptibility  to  the  particular  pathogenic 
organism  it  is  desired  to  inoculate.  Indeed,  with  some 
of  the  more  sensitive  and  strictly  parasitic  bacteria  this 
method  of  animal  inoculation  is  practically  the  only 
method  that  will  yield  a  satisfactory  result. 


XVI.  METHODS  OF  IDENTIFICATION 
AND  STUDY. 

IN  order  to  identify  an  organism  after  isolation, 
tube,  plate,  and  other  cultivations  must  be  prepared, 
incubated  under  suitable  conditions  as  to  temperature 
and  environment,  and  examined  from  time  to  time 
(a)  macroscopically,  (b)  by  microscopical  methods,  (c) 
by  chemical  methods,  (d)  by  physical  methods,  (e) 
by  inoculation  methods,  and  the  results  of  these  exami- 
nations duly  recorded. 

It  must  be  stated  definitely  that  no  micro-organism 
can  be  identified  by  any  one  character  or  property, 
whether  microscopical,  biological  or  chemical,  but 
that  on  the  contrary  its  entire  life  history  must  be 
carefully  studied  and  then  its  identity  established 
from  a  consideration  of  the  sum  total  of  these 
observations. 

In  order  to  give  to  the  recorded  results  their  maxi- 
mum value  it  is  essential  that  they  should  be  exact 
and  systematic,  therefore  some  such  scheme  as  the 
following  should  be  adhered  to;  and  especially  is  this 
necessary  in  describing  an  organism  not  previously 
isolated  and  studied. 

SCHEME  OF  STUDY. 
Designation  : 

Originally  isolated  by  (observer's  name)  in  (date), 
from  (source  of  organism)  . 

1.  Cultural     Characters.  —  (Vide  Macroscopical    Ex- 

amination of  Cultivation,  page  261.) 
Gelatine  plates, 


Gelatine  streak, 

O    1     x-  t    U  a      20 

Gelatine  stab, 
Gelatine  shake, 

259 


26o  METHODS    OF    IDENTIFICATION   AND    STUDY 

Agar  plates, 

Agar  streak  or  smear, 

Agar  stab, 


Inspissated  blood-serum, 


at2o°C.  and  3  7°  C. 


Bouillon, 
Litmus  milk, 
Potato, 

Special  media  for  the  purpose  of  demonstrating 
characteristic  appearances. 

2.  Morphology. — (Vide  Microscopical  Examination  of 

Cultivations,  page  272.) 
Vegetative  forms : 

Shape. 

Size. 

Motility. 

Flagella  (if  present) . 

Capsule  (if  present) . 

Involution  forms. 

Pleomorphism  (if  observed) . 
Sporing  forms   (if  observed).     Of  which  class? 
Staining  reactions. 

3.  Chemical    Products  of  Growth. — (Vide  Chemical 

Examination  of  Cultivations,  page  276.) 
Chromogenesis . 
Photogenesis. 
Enzyme  formation. 

Fermentation  of  carbohydrates : 
Acid  formation. 
Alkali  formation. 
Indol  formation. 
Phenol  formation. 
Reducing  and  oxidising  substances. 
Gas  formation. 

4.  Biology. — (Vide  Physical  Examination  of  Cultures, 

page  295.) 
Atmosphere. 
Temperature. 


PLATE    CULTURES  26l 

Reaction  of  nutrient  media. 
Resistance  to  lethal  agents : 
Physical : 
Desiccation. 
Light. 
Colours. 
Chemical  germicides. 
Vitality. 
5.  Pathogenicity : 

Susceptible  animals,  subsequently  arranged  in 

order  of  susceptibility. 
Immune  animals. 

Experimental  inoculation,  symptoms  of  disease. 
Post-mortem  appearances. 
Virulence : 

Length  of  time  maintained. 
Optimum  medium? 
Minimal  lethal  dose. 

Exaltation  and  attenuation  of  virulence?     . 
Toxin  formation. 

MACROSCOPICAL  EXAMINATION  OF  CULTIVATIONS. 

In  describing  the  naked-eye  and  low-power  appear- 
ances of  the  bacterial  growth  the  descriptive  terms 
introduced  by  Chester  (and  included  in  the  following 
scheme)  should  be  employed. 

SOLID  MEDIA. 

Plate  Cultures.— 

Gelatine. — Note  the  presence  or  absence  of  lique- 
faction of  the  surrounding  medium.  If  liquefaction 
is  present,  note  shape  and  character  (vide  page  269, 
"stab"  cultures). 

Agar. — No  liquefaction  takes  place  in  this  medium. 
The  liquid  found  on  the  surface  of  the  agar  (or  at  the 
bottom  of  the  tube  in  agar  tube  cultures)  is  merely 


262  METHODS    OF   IDENTIFICATION   AND    STUDY 

water  which  has  been  expressed  during  the  rapid  solidi- 
fication of  the  medium  and  has  subsequently  condensed. 
Gelatine  and  Agar. — Examine  the  colonies  at  inter- 
vals of  twenty-four  hours. 

(a)  With  the  naked  eye. 

(b)  With  a  hand  lens  or  watchmaker's  glass. 

(c)  Under  a  low  power  (i  inch)  of  the  microscope, 
or  by  means  of  a  small  dissecting  microscope. 

Distinguish  superficial  from  deep  colonies  and  note 
the  characters  of  the  individual  colonies. 

(A )  Size. — The  diameter  in  millimetres ,  at  the  various 
ages. 

(B)  Shape.— 

Punctiform:  Dimensions  too  slight  for  defining  form 
by  naked  eye;  minute,  raised,  hemispherical. 


b  c 

FIG.  141. — Types  of  colonies:  a,  Cochleate;  6,  amoeboid;  c,  mycelioid. 

Round:  Of  a  more  or  less  circular  outline. 

Elliptical :  Of  a  more  or  less  oval  outline. 

Irregular :  Outlines  not  conforming  to  any  recognised 
shape. 

Fusiform:  Spindle-shaped,  tapering  at  each  end. 

Cochleate:  Spiral  or  twisted  like  a  snail  shell  (Fig. 
141,  a). 


'SURFACE  ELEVATIONS  263 

Amoeboid:  Very  irregular,  streaming  (Fig.   141,  6). 

Mycelioid :  A  filamentous  colony,  with  the  radiate 
character  of  a  mould  (Fig.  141,  c). 

Filamentous :  An  irregular  mass  of  loosely  woven 
filaments  (Fig.  142,  a). 

Floccose:  Of  a  dense  woolly  structure. 

Rhizoid:  Of  an  irregular,  branched,  root-like  char- 
acter (Fig.  142,  b). 

Conglomerate :  An  aggregate  of  colonies  of  similar 
size  and  form  (Fig.  142,  c). 


FIG.  142. — Types  of  colonies:  a,  Filamentous;  b,  rhizoid;  c,  conglomerate; 

d,  toruloid. 

Toruloid :  An  aggregate  of  colonies,  like  the  budding 
of  the  yeast  plant  (Fig.  142,  d). 

Rosulate :  Shaped  like  a  rosette. 

(C)  Surface  Elevation. — 

i .  General  Character  of  Surface  as  a  Whole: 

Flat :  Thin,  leafy,  spreading  over  the  surface  (Fig. 
143,  a). 

Effused:  Spread  over  the  surface  as  a  thin,  veily 
layer,  more  delicate  than  the  preceding. 

Raised:  Growth  thick,  with  abrupt  terraced  edges 
(Fig.  143,  b). 

Convex:  Surface  the  segment  of  a  circle,  but  very 
flatly  convex  (Fig.  143,  c). 


264 


METHODS    OF   IDENTIFICATION   AND    STUDY 


Pulvinate:  Surface  the  segment  of  a  circle,  but  de- 
cidedly convex  (Fig.  143,  d). 

Capitate:  Surface  hemispherical  (Fig.  143,  e). 
Umbilicate :  Having  a  central  pit  or  depression  (Fig. 

143, /)• 

Conical:  Cone  with  rounded  apex  (Fig.  143,  g). 

Umbonate:  Having  a  central  con- 
vex nipple-like  elevation  (Fig.  143,  h). 

2 .  Detailed  Characters  of  Surface: 

Smooth :  Surface  even,  without  any 
of  the  following  distinctive  characters. 

Alveolate:  Marked  by  depressions 
separated  by  thin  walls  so  as  to  re- 
semble a  honeycomb  (Fig.  144). 

Punctate:  Dotted  with  punctures 
like  pin-pricks. 

Bullate:  Like  a  blistered  surface, 
rising  in  convex  prominences,  rather 
coarse. 

Vesicular:    More    or    less   covered 


FIG.  143.  FIG.  144. 

FIG.  143. — Surface   elevation  of  colonies:  a,  Flat;  &,  raised;  c,  convex; 
d,  pulvinate;  e,  capitate;  /,  umbilicate;  g,  conical;  h,  umbonate. 
FIG.  144. — Types  of  colonies — alveolate. 

with  minute  vesicles  due  to  gas  formation;  more 
minute  than  bullate. 

Verrucose:  Wart-like,  bearing  wart-like  prominences. 

Squamose:  Scaly,  covered  with  scales. 

Echinate:  Beset  with  pointed  prominences. 

Papillate:  Beset  with  nipple  or  mamma-like  proc- 
esses. 


INTERNAL  STRUCTURE  OF  COLONY         265 

Rugose:  Short  irregular  folds,  due  to  shrinkage  of 
surface  growth. 

Corrugated .  In  long  folds,  due  to  shrinkage. 

Contoured:  An  irregular  but  smoothly  undulating 
surface,  resembling  the  surface  of  a  relief  map. 

Rimose :  Abounding  in  chinks,  clefts,  or  cracks. 

(D)  Internal  Structure  of  Colony  (Microscopical).— 
Refraction  Weak :  Outline  and  surface  of  relief  not 

strongly  defined. 

Refraction    Strong:    Outline   and   surface   of   relief 

strongly  defined;  dense,  not  filamentous  colonies. 


a  b  c 

FIG.  145. — Types  of  colonies:  a,  Grumose;  6,  moruloid;  c,  clouded. 

1.  General: 

Amorphous :  Without  any  definite  structure,  such  as 
is  specified  below. 

Hyaline :  Clear  and  colourless. 

Homogeneous:  Structure  uniform  throughout  all 
parts  of  the  colony. 

Homochromous :  Colour  uniform  throughout. 

2.  Granulations  or  Blotchings: 
Finely  granular. 

Coarsely  granular. 

Grumose :  Coarser  than  the  preceding,  with  a  clotted 


266 


METHODS    OF   IDENTIFICATION   AND    STUDY 


appearance,    and  particles   in   clustered   grains    (Fig. 

145.°)- 

Moruloid:  Having  the  character  of  a  mulberry,  seg- 
mented, by  which  the  colony  is  divided  in  more  or 
less  regular  segments  (Fig.  145,  b). 

Clouded:  Having  a  pale  ground,  with  ill-defined 
patches  of  a  deeper  tint  (Fig.  145,  c). 


a  b  c 

FIG.  146. — Types  of  colonies:  a,  Reticulate;  b,  gyrose;  c,  marmorated. 

3.  Colony  Marking  or  Striping: 

Reticulate:  In  the  form  of  a  network,  like  the  veins 
of  a  leaf  (Fig.  146,  a). 

Areolate:  Divided  into  rather  irregular,  or  angular, 
spaces  by  more  or  less  definite  boundaries. 

Gyrose:  Marked  by  wavy  lines, 
indefinitely  placed  (Fig.  146,  b). 

Marmorated:  Showing  faint,  ir- 
regular stripes,  or  traversed  by 
vein-like  markings,  as  in  marble 
(Fig.  146,  c). 

Rivulose:  Marked  by  lines  like 
the  rivers  of  a  map. 

Rimose :  Showing  chinks,  cracks, 
or  clefts. 

4.  Filamentous  Colonies: 
Filamentous :  As  already  defined. 

Floccose :  Composed  of  filaments,  densely  placed. 


FIG.  147. — Types  of 
colonies — curled . 


OPTICAL    CHARACTERS  267 

Curled:  Filaments  in  parallel  strands,  like  locks  or 
ringlets  (Fig.  147). 

(E)  Edges  of  Colonies. — 

Entire:  Without  toothing  or  division  (Fig.  148,  a). 

Undulate:  Wavy  (Fig.  148,  b). 

Repand:  Like  the  border  of  an  open  umbrella  (Fig. 
148,  c). 

Erose:  As  if  gnawed,  irregularly  toothed  (Fig.  148,  d). 


FIG.  148. — Edges  of  colonies:  a,  Entire;  6,  undulate;  c,  repand;  d,  erose. 

Lobate. 

Lobulate:  Minutely  lobate  (Fig.  149,  e). 
Auriculate :  With  ear-like  lobes  (Fig.  1 49,  f) . 
Lacerate:  Irregularly  cleft,  as  if  torn  (Fig.  149,  g) 
Fimbriate:  Fringed  (Fig.  149,  h). 
Ciliate:  Hair-like  extensions,  radiately  placed  (Fig. 
149, /)• 


FIG.  149. — Edges  of  colonies:  e,  Lobar-lobulate;  /,  auriculate;  g,  lacerate; 
h,  fimbriate;  i,  ciliate. 

Tufted. 

Filamentous :  As  already  defined. 

Curled :  As  already  defined. 

(F)  Optical  Characters  (after  Shuttleworth)  .— 

i.  General  Characters: 

Transparent :  Transmitting  light. 


268  METHODS    OF   IDENTIFICATION   AND    STUDY 

Vitreous :  Transparent  and  colourless. 

Oleaginous:  Transparent  and  yellow;  olive  to  lin- 
seed-oil coloured. 

Resinous :  Transparent  and  brown,  varnish  or  resin- 
coloured. 

Translucent:  Faintly  transparent. 

Porcelaneous :  Translucent  and  white. 

Opalescent:  Translucent;  greyish- white  by  reflected 
light. 

Nacreous:  Translucent,  greyish-white,  with  pearly 
lustre. 

Sebaceous:  Translucent,  yellowish  or  greyish- white. 

Butyrous :  Translucent  and  yellow. 

Ceraceous:  Translucent  and  wax-coloured. 

Opaque. 

Cretaceous :  Opaque  and  white,  chalky. 

Dull :  Without  lustre. 

Glistening:  Shining. 

Fluorescent. 

Iridescent. 

2.  Chromogenicity: 

Colour  of  pigment. 

Pigment  restricted  to  colonies. 

Pigment  restricted  to  medium  surrounding  colonies. 

Pigment  present  in  colonies  and  in  medium. 

Streak  or  Smear  Cultures.— 

Gelatine  and  Agar. — Note  general  points  as  indicated 
under  plate  cultivations. 

Inspissated  Blood-serum. — Note  the  presence  or 
absence  of  liquefaction  of  the  medium.  (The  presence 
of  condensation  water  at  the  bottom  of  the  tube  must 
not  be  confounded  with  liquefaction  of  the  medium.) 

All  Oblique  Tube  Cultures — 

i.  Colonies  Discrete:  Size,  shape,  etc.,  as  for  plate 
cultivations  (vide  page  261). 


GELATINE    STAB    CULTURES  269 

2.  Colonies  Confluent:  Surface  elevation  and  char- 
acter of  edge,  as  for  plate  cultivations  (vide  page  263). 
Chromogenicity:  As  for  plate  cultures. 

Gelatine  Stab  Cultures. — 

(A)  Surface  Growth. — As  for  individual  colonies  in 
plate  cultures  (vide  page  261). 


FIG.  150. — Stab  cultivations — types  of  growth:  a,  Filiform;  b,  beaded; 
c,  echinate;  d,  villous;  e,  arborescent. 

(B)  Line  of  Puncture.— 

Filiform:    Uniform    growth,    without    special    char- 
acters (Fig.  150,  a). 

Nodose :  Consisting  of  closely  aggregated  colonies. 


270 


METHODS    OF   IDENTIFICATION    AND    STUDY 


Beaded:  Consisting  of  loosely  placed  or  disjointed 
colonies  (Fig.  150,  b). 

Papillate:  Beset  with  papillate  extensions. 

Echinate:  Beset  with  acicular  extensions  (Fig.  150,  c). 

Villous:  Beset  with  short,  undivided,  hair-like 
extensions  (Fig.  150,  d). 

Plumose:  A  delicate  feathery  growth. 


FIG.  151. — Stab  cultivations — types  of  growth:  /,  Crateriform;  g,  saccate; 
h,   infundibuliform;  ;,   napiform;   k,  fusiform;   /,   stratiform. 

Arborescent:    Branched    or    tree-like,    beset    with 
branched  hair-like  extensions  (Fig.  150,  e). 
(Q  Area  of  Liquefaction  (if  present)  .— 
Crateriform:   A   saucer-shaped   liquefaction   of   the 
gelatine  (Fig.  151,7). 


FLUID    MEDIA  271 

Saccate:  Shape  of  an  elongated  sack,  tubular  cylin- 
drical (Fig.  151,  g). 

Infundibuliform :   Shape  of  a  funnel,   conical    (Fig. 

IS1*  *0- 

Napiform:  Shape  of  a  turnip  (Fig.  151,  /). 

Fusiform :  Outline  of  a  parsnip,  narrow  at  either  end, 
broadest  below  the  surface  (Fig.  151,  k). 

Stratiform:  Liquefaction  extending  to  the  walls  of 
the  tube  and  downward  horizontally  (Fig.  151,  /). 

(D)  Character  of  the  Liquefied  Gelatine. — 

1.  Pellicle  on  surface. 

2.  Uniformly  turbid. 

3.  Granular. 

4.  Mainly  clear,  but  containing  flocculi. 

5.  Deposit  at  apex  of  liquefied  portion. 

(E)  Production  of  Gas  Bubbles. 

Shake  Cultures. — 

1.  Presence  or  absence  of  liquefaction. 

2.  Production  of  gas  bubbles. 

3 .  Bulk  of  growth  at  the  surface — aerobic. 

4.  Bulk  of  growth  in  depths — anaerobic. 

Fluid  Media. 

1 .  Surface  of  the  Liquid.— 

Presence  or  absence  of  froth  due  to  gas  bubbles. 
Presence  or  absence  of  pellicle  formation. 
Character  of  pellicle. 

2.  Body  of  the  Liquid.— 

Uniformly  turbid. 

Flocculi  in  suspension. 

Granules  in  suspension. 

Clear,  with  precipitate  at  bottom  of  tube. 

Colouration  of  fluid,  presence  or  absence  of. 

3.  Precipitate. — 

Character. 


272  METHODS    OF   IDENTIFICATION  AND    STUDY 

Amount. 
Colour. 

Carbohydrate  Media. — 

Growth. 
Reaction. 
Gas  formation. 

Coagulation  or  not  of  serum  albumen  (when  serum 
water  media  are  employed). 

Litmus  Milk  Cultivations.— 

Unaltered. 


Reaction : 


Acid. 


Alkaline. 

2.  Odour. 

3.  Formation  of  gas. 

Unaltered. 


4.  Consistency: 


Peptonised  (character  of  solution) . 


Coagulated. 

f  hard:  solid. 

soft :  floculent. 
5.  Clot:  Character  ,        ,  ,      -  u 

ragged  and  broken  up  by  gas 

(      bubbles. 

(a)  Coagulum  undissolved. 

(b)  Coagulum  finally  peptonised,  completely :  in- 

completely. 
Resulting  solution,  clear:  turbid. 

Abundant. 
Scanty. 


6.  Whey 


Clear. 

Turbid. 

Coagulated  by  boiling,  or  not. 


BY  MICROSCOPICAL  METHODS. 

As   a   council   of   perfection   preparations  must  be 
made  from  pure  cultivations  4,  6,  8,  12,  18,  and  24 


LITMUS    MILK   CULTIVATIONS  273 

hours;  and  subsequently  at  intervals  of,  say,  twenty- 
four  hours,  during  the  entire  period  they  are  under 
observation,  and  examined — 

(A)  Living. — 1.  In  hanging  drop,  to  determine  mo- 
tility  or  non-motility. 

In  this  connection  it  must  be  remembered  that 
under  certain  conditions  as  to  environment  (e.  g., 
when  examined  in  an  unsuitable  medium,  atmosphere, 
temperature,  etc.)  motile  bacilli  may  fail  to  exhibit 
activity.  No  organism,  therefore,  should  be  recorded 
as  non-motile  from  one  observation  only;  a  series  of 
observations  at  different  ages  and  under  varying  con- 
ditions should  form  the  basis  of  an  opinion  as  to  the 
absence  of  true  locomotion. 

Size. — In  the  case  of  non-motile  or  sluggishly  motile 
organisms,  endeavour  to  measure  several  individuals 
in  each  hanging  drop  by  means  of  the  eyepiece  microm- 
eter or  the  eikonometer  (vide  page  63),  and  average  the 
results. 

If  the  organism  is  one  which  forms  spores,  observe — 

(a)  Spore  Formation. — Prepare  hanging-drop  culti- 
vations (vide  page  78)  from  vegetative  forms  of  the 
organism,  adding  a  trace  of  magenta  solution  (0.5  per 
cent.)  or  other  intra  vitam  stain  (see  page  77)  to  the 
drop,  on  the  point  of  the  platinum  needle,  to  facilitate 
the  observation  of  the  phenomenon  by  rendering  the 
bacilli  more  distinct. 

Place  the  preparation  on  the  stage  of  the  micro- 
scope ;  if  necessary,  using  a  warm  stage. 

Arrange  illumination,  etc.,  and  select  a  solitary 
bacillus  for  observation,  by  the  help  of  the  |-inch  lens. 

Substitute  the  -^-inch  oil-immersion  lens  for  the 
sixth,  and  observe  the  formation  of  the  spore;  if 
possible,  measure  any  alteration  in  size  which  may 
occur  by  means  of  the  Ramsden  micrometer. 

(b)  Spore  Germination. — Prepare  hanging-drop  culti- 
vations from  old  cultivations  in  which  no  living  vegeta- 

18 


274  METHODS    OF   IDENTIFICATION    AND    STUDY 

tive  forms  are  present,  and  observe  the  process  of 
germination  in  a  similar  manner. 

The  comfort  of  the  microscopist  is  largely  enhanced 
in  those  cases  where  the  period  of  observation  is  at  all 
lengthy,  by  use  of  some  form  of  eye  screen  before  the 
unemployed  eye,  such  as  is  figured  on  page  58  (Fig.  49). 

If  it  is  impossible  to  carry  out  the  method  suggested 
above,  proceed  as  follows : 

(a)  Spore  Formation. — Plant  the  organism  in  broth 
and  incubate  under  optimum  conditions. 

At  regular  intervals,  say  every  thirty  minutes,  re- 
move a  loopful  of  the  cultivation  and  prepare  a  cover- 
slip  film  preparation. 

Fix,  while  still  wet,  in  the  corrosive  sublimate  fixing 
solution. 

Stain  with  aniline  gentian  violet,  and  partially  de- 
colourise with  2  per  cent,  acetic  acid. 

Mount  and  number  consecutively ;  then  examine. 

(b)  Spore  Germination. — Expose  a  thick  emulsion  of 
the  spores  to  a  temperature  of  80°  C.  for  ten  minutes 
in  the  differential  steriliser  (vide  page  257). 

Transfer  the  emulsion  to  a  tube  of  sterile  nutrient 
broth  and  incubate. 

Remove  specimens  from  the  tube  culture  at  intervals 
of,  say,  five  minutes. 

Fix,  stain,  etc.,  wet,  as  under   (a),  and  examine. 

(B)  Fixed. — 2.  In  stained  preparations. 

(a)  To  determine  points  in  morphology: 

Shape  (vide  classification,  page  131). 

Size: 

(a)  Prepare  cover-slip  film  preparations  at  the 
various  ages,  and  fix  by  exposure  to  a  tem- 
perature of  115°  C.  for  twenty  minutes  in  hot- 
air  oven. 

(b)  Stain  the   preparations   by  Gram's  method 
(if  applicable)  or  with  dilute  carbol-fuchsin, 
and  mount  in  the  usual  way. 


LITMUS    MILK    CULTIVATIONS  275 

(c)  Measure  (vide  page  66)  some  twety-five  indi- 
viduals in  each  film  by  means  of  the  Ramsden  's 
or   the   stage   micrometer   and   average   the 
result. 
Pleomorphism;  If  noted,  record — 

The  predominant  character  of  the  variant  forms. 

On  what  medium  or  media  they  are  observed. 

At  what  period  of  development. 
(b)  To  demonstrate  details  of  structure: 
Flagella:  If  noted,  record — 

Method  of  staining  (vide  page  101). 

Position    and    arrangement    (vide    page    136). 

Number. 
Spores:  If  noted,  record — 

Method  of  staining. 

Shape. 

Size. 

Position  within  the  parent  cell. 

Condition,  as  to  shape,  of  the  parent  cell  (vide 
page  139). 

Optimum  medium  and  temperature. 

Age  of  cultivation. 

Conditions  of  environment  as  to  temperature, 
atmosphere. 

Method  of  germination   (vide  page   140). 
Involution  Forms:  If  noted,  record — 

Method  of  staining. 

Character  (e.  g.t  if  living  or  dead). 

Shape. 

On  what  medium  they  are  observed. 

Age  of  medium. 

Environment. 
Metachromatic  Granules:  If  noted,  record — 

Method  of  staining. 

Character  of  granules. 

Number  of  granules. 

Colour  of  granules. 


276  METHODS    OF   IDENTIFICATION  AND   STUDY 

3.  Staining  Reactions. — 

1.  Gram's  Method. — Positive  or  negative. 

2.  Neisser's  Method. — If  granules  are  noted,  record — 

1.  Position. 

2.  Number. 

3 .  Ziehl-Neelsen's  Method. — Acid-fast  or  decolourised. 

4.  Simple  Aniline  Dyes. — (Noting  those  giving  the 
best  results,  with  details  of  staining  processes.) 

Methylene-blue 

Fuchsin  ...-     ,. 

~     , .         .  1  ,        and  their  modifications. 
Gentian  violet 

Thionine  blue 

BY  BIOCHEMICAL  METHODS. 

Test  cultivations  of  the  organism  for  the  presence  of — 

Soluble    enzymes — proteolytic,    diastatic,  invertase. 

Organic  acids — (a)  quantitatively — i.  e.,  estimate 
the  total  acid  production;  (b)  qualitatively  for  formic, 
acetic,  propionic,  butyric,  lactic. 

Ammonia. 

Neutral  volatile  substances — ethyl  alcohol,  aldehyde, 
acetone. 

Aromatic  products — indol,  phenol. 

Soluble  pigments. 

Test  the  power  of  reducing  (a)  colouring  matters, 
(b)  nitrates  to  nitrites. 

Investigate  the  gas  production — H2S,  CO2,H2.  Esti- 
mate the  ratio  between  the  last  two  gases. 

Prepare  all  cultivations  for  these  methods  of  ex- 
amination under  optimum  conditions,  previously  deter- 
mined for  each  of  the  organisms  it  is  intended  to  investi- 
gate, as  to 

(a)  Reaction  of  medium ; 

(b)  Incubation  temperature; 

(c)  Atmospheric  environment; 


ENZYME    PRODUCTION  277 

and  keep  careful  records  of  these  points,  and  also  of  the 
age  of  the  cultivation  used  in  the  final  examination. 

Examine  the  cultivations  for  the  various  products  of 
bacterial  metabolism  after  forty-eight  hours'  growth, 
and  never  omit  to  examine  "control"  (uninoculated) 
tube  or  flask  of  medium  from  the  same  batch,  kept  for 
a  similar  period  under  identical  conditions. 

If  the  results  are  negative,  test  further  cultivations 
at  three  days,  five  days,  and  ten  days. 

1.  Enzyme  Production. — 

(A)  Proteolytic  Enzymes. — (Convert  proteins  into 
proteose,  peptone  and  further  products  of  hydrolysis; 
e.  g.,  B.  pyocyaneus.) 

Media  Required: 

Blood-serum  and  milk-serum  which  have  been  carefully  filtered 
through  a  porcelain  candle. 
Reagents  Required: 

Ammonium  sulphate. 

Thirty  per  cent,  caustic  soda  solution. 

Copper  sulphate,  0.5  per  cent,  aqueous  solution. 

One  per  cent,  acetic  acid  solution. 

Millon's  reagent. 

Glyoxylic  acid  solution. 

Concentrated  sulphuric  acid. 

METHOD.— 

1.  Prepare  cultivations  in  bulk  (50  c.c.)  in  a  flask 
and  incubate. 

2.  Make  the  liquid  faintly  acid  with  acetic  acid,  then 
boil.     (This  precipitates  the  unaltered  proteins.) 

3.  Filter. 

4.  Take  10  c.c.  of  the  filtrate  in  a  test-tube  and  add 
i  c.c.  of  the  caustic  soda,  then  add  the  copper  sulphate 
drop  by  drop. 

Pink  colour  which  becomes  violet  with  more 
copper    sulphate    =    proteose  and  peptone. 

5.  Saturate  the  rest  of  the  filtrate  with  ammonium 
sulphate. 

Precipitate  =  proteose. 


278  METHODS    OF   IDENTIFICATION    AND    STUDY 

6.  Filter  and  divide  the  filtrate  into  three  parts  a,  b 
and  c. 

a.  Repeat  the  copper  sulphate  test,  using  excess  of 
caustic  soda  to  displace  the  ammonia  from  the  am- 
monium sulphate. 

Pink  colour  =  peptone. 

b.  Boil  with  Millon's  reagent. 
Red  colour  =  tyrosine. 

c.  Add  glyoxylic  acid  solution  and  run  in  concen- 
trated sulphuric  acid. 

Violet  ring  at  upper  level  of  acid  =   tryptophane. 

Both  the  tyrosine  and  tryptophane  may  be  either 
in  the  free  state  or  in  combination  as  polypeptid  or 
peptone. 

(B)  Diastase. — (Converts  starch  into  sugar;  e.g.,  B. 
subtilis.) 

Medium  Required: 

Inosite-free  bouillon. 
Reagents  Required: 

Starch. 

Thymol. 

Fehling's  soltuion. 

METHOD.— 

1.  Prepare  tube  cultivation  and  incubate. 

2.  Prepare  a  thin  starch  paste  and  add  2  per  cent, 
thymol  to  it. 

3.  Mix  equal  parts  of  the  cultivation  to  be  tested 
and  the  starch  paste,  and  place  in  the  incubator  at  3  7° 
C.  for  six  to  eight  hours. 

4.  Filter. 

Test  the  filtrate  for  sugar. 

Boil  some  of  the  Fehling's  solution  in  a  test-tube. 

Add  the  nitrate  drop  by  drop  until,  if  necessary,  a 
quantity  has  been  added  equal  in  amount  to  the  Feh- 
ling's solution  employed,  keeping  the  mixture  at  the 
boiling-point  during  the  process. 

Yellow  or  orange  precipitate  =  sugar. 


FERMENTATION    REACTION  279 

(C)  Invertase. — (Convert  saccharose  into  a  mixture  of 
dextrose  and  laevulose  e.g.,  B.  fluorescens  liquefaciens.) 

Medium  Required: 

Inosite-free  bouillon. 
Reagents  Required: 

Cane  sugar,  2  per  cent,  aqueous  solution. 

Carbolic  acid. 

METHOD. — 

1.  Prepare  tube  cultivations  and  incubate. 

2.  Add  2  per  cent,  of  carbolic  acid  to  the  sugar 
solution. 

3.  Mix  equal  quantities  of  the  carbolised  sugar  solu- 
tion  and   the   cultivation   in   a   test-tube;  allow   the 
mixture  to  stand  for  several  hours. 

4.  Filter. 

,  Test  the  filtrate  for  reducing  sugar  as  in  the   pre- 
ceding section. 

(D)  Rennin  and  ''Lab'''  Enzymes. — (Coagulate  milk 
independently  of  the  action  of  acids;  e.  g.,   B.  pro- 
digiosus.) 

Media  Required: 

Inosite-free  bouillon. 
Litmus  milk. 

METHOD. — 

1.  Prepare  tube  cultivations  and  incubate. 

2.  After  incubation  heat  the  cultivation  to  55°  C. 
for  half  an  hour,  to  sterilise. 

3.  By  means  of  a  sterile  pipette  run  5  c.c.  of  the 
cultivation  into  each  of  three  tubes  of  litmus  milk. 

4.  Place  in  the  cold  incubator  at  22°  C.  and  examine 
each  day  for  ten  days. 

Absence  of  coagulation  at  the  end  of  that  period 
will  indicate  absence  of  rennin  ferment  formation. 

Fermentation  Reactions. 

As  tested  upon  carbohydrate  substances  and  organic 
salts. 


280  METHODS    OF   IDENTIFICATION  AND    STUDY 

Media  Required: 

Peptone  water  containing  various  percentages  (gen- 
erally 2  per  cent.)  of  each  of  the  substances  referred 
to  under  "sugar"  media  (page  177),  also  tubes  of 
peptone  water  containing  i  per  cent,  respectively  of 
each  of  the  following : 

Organic  salts:  Sodium  citrate,  formate,  lactate,  malate, 

tartrate. 

METHOD. — 

1.  Prepare  tube  cultivations  in   each  of  the  above 
media. 

2.  Observe  from  day  to  day  up  to  the  expiration  of 
ten  days  if  necessary. 

3.  Note  growth,  reaction,  gas  production. 

2.  Acid  Production. 

(a)   Quantitative. — 
Medium  Required: 

Sugar  (glucose)  bouillon  of  known  "optimum"  reaction. 
Apparatus  and  Reagents  Required: 

As  for  estimating  reaction  of  media  (vide  page  150). 

METHOD.— 

1.  Prepare  cultivation  in  bulk  (100  c.c.)  in  a  flask; 
also  "control"  flask  of  medium  from  same  batch. 

2.  After  suitable  incubation,  heat  both  flasks  in  the 
steamer  at  100°  C.  for  thirty  minutes  to  sterilise. 

3 .  Determine  the  litre  of  the  medium  in  "  inoculated  " 
and  "control"  flasks  as  described  in  the  preparation  of 
nutrient  media  (vide  page  151). 

4.  The  difference  between  the  titre  of  the  medium 
in  the  two  flasks  gives  the  total  acid  production  of  the 
bacterium  under  observation  in  terms  of  normal  NaOH. 

NOTE. — If  the  growth  is  very  heavy  it  may  be  a  difficult  matter 
to  determine  the  end-point.  The  cultivation  should  then  be 
filtered  through  a  Berkfeld  filter  candle  previous  to  step  2,  and 
the  filtrate  employed  in  the  titration. 


ACID     PRODUCTION  281 

(&)   Qualitative  (of  all  the  organic  acids  present). — 
Medium  Required: 

Sugar  (glucose  or  lactose)  bouillon  as  in  quantitative  examina- 
tion. 
Reagents  Required: 

Hydrochloric  acid,  concentrated. 

Hydrochloric  acid,  25  per  cent. 

Sulphuric  acid,  concentrated  (pure). 

Phosphoric  acid,  concentrated  solution. 

Ammonia. 

Ammonium  sulphate. 

Baryta  water. 

Sodium  carbonate,  saturated  aqueous  solution. 

Absolute  alcohol. 

Ether. 

Calcium  chloride. 

Calcium  chloride  solution. 

Zinc  carbonate. 

Copper  sulphate  saturated  aqueous  solution. 

Alcoholic  thiophene  solution  (0.15  c.c.  in  100  c.c.). 

Animal  charcoal. 

Five  per  cent,  sodium  nitroprusside  solution. 

Potassium  bichromate. 

Scruff's  reagent. 

Arsenious  oxide. 

Ferric  chloride,  4  per  cent,  aqueous  solution. 

Silver  nitrate,   i  per  cent,  aqueous  solution. 

Lugol's  iodine. 

Ten  per  cent,  caustic  soda  solution. 

Hard  paraffin  wax  (melting-point  about  52°  C.). 

METHOD. — • 

1.  Prepare  cultivation  in  bulk  (500  c.c.)  in  a  litre 
flask  and  add  sterilised  precipitated  chalk,  10  grammes. 
Incubate  at  the  optimum  temperature. 

2.  After  incubation  throw  a  piece  of  paraffin  wax 
(about  a  centimetre  cube)   into  the  cultivation  and 
connect  up  the  flask  with  a  condenser. 

The  paraffin,  which  liquefies  and  forms  a  thin  layer 
on  the  surface  of  the  fluid,  is  necessary  to  prevent 
the  cultivation  frothing  up  and  running  unaltered 
through  the  condenser  during  the  subsequent  process 
of  distillation. 

3.  Distill  over  200  to  300  c.c. 


282 


METHODS    OF   IDENTIFICATION   AND    STUDY 


Use  a  rose-top  burner  to  minimise  the  danger  of 
cracking  the  flask;  and  to  the  same  end,  well  agitate 
the  contents  of  the  flask  to  prevent  the  chalk  settling. 

The  distillate  "A"  will  contain  alcohol,  etc.  (vide 
page  285) ;  the  residue  "a"  will  contain  the  volatile 
and  fixed  acids. 

4.  Disconnect  the  flask  and  filter.  The  residue  "a" 
then  =  filtrate  B  and  residue  b. 


v 

FIG.  152. — Arrangement  of  distillation  apparatus  for  acids,  etc. 

5 .  Residue  b.     Wash  the  residue  from  the  filter  paper, 
dissolve  by  heating  with  dilute  hydrochloric  acid,  and 
add    calcium    chloride    solution    and    ammonia   until 
alkaline. 

White  precipitate  insoluble  in  acetic  acid  =  oxalic 
acid. 

6.  Make  up  filtrate  B  to  500  c.c.  with  distilled  water 
and  divide  into  two  parts. 


ACID    PRODUCTION 


283 


7.  Acidify  250  c.c.  with  20  c.c.  concentrated  phos- 
phoric acid  (this  liberates  the  volatile  acids)  and  distil 
to  small  bulk. 

The  distillate  "B"  may  contain  formic,  acetic, 
propionic,  butyric  and  benzoic  acids. 

DISTILLATE  "  B." 
(Volatile  Acids.) 


i.  Add  baryta  water  till  alkaline, 
and  evaporate  to  dryness. 

2.  Add  50  c.c.  absolute  alcohol  and  allow 

to  stand,  with  frequent  stirring,  for 
two  to  three  hours. 

3.  Filter  and  wash  with  alcohol. 


FILTRATE 

may  contain  barium  propionate, 
barium  butyrate. 


1.  Evaporate  to  dryness. 

2.  Dissolve  residue  in  150  c.c.  water. 

3.  Acidify  with  phosphoric  acid  and  dis- 

til. 

4.  Saturate  distillate  with  calcium  chlo- 

ride and  distill  over  a  few  c.c. 

5.  Test  distillate  for  butyric  acid: 

Add  3  c.c.  alcohol  and  4  drops  con- 
centrated sulphuric  acid. 
Smell  of  pineapple  =  butyric  acid. 
Propionic     acid    in    small    quantities 
cannot  be  distinguished  from  buty- 
ric acid    by  tests    within  the  scope 
of   the  bacteriological  laboratory. 


RESIDUE 

may  contain  barium  acetate, 
barium  formate,  barium  benzoate. 


1.  Evaporate  off  alcohol  and  dissolve 

up  the  residue  on  the  filter  in  hot 
water  and  neutralise. 

2.  Divide  the  solution  into  four  por- 

tions : 

(a)  Add  ferric  chloride  solution. 
Brown  colour  =  acetic  or  for- 
mic acids. 

Buff  ppt.  =  benzoic  acid   (see 
ether  soluble  acids). 

(b)  Add   silver  nitrate   solution; 

then   add    one  drop    am- 
monia water,  and  boil. 
Black     precipitate    of    me- 
tallic silver  —formic  acid. 

(c)  Evaporate    to    dryness;  mix 

with    equal    quantity    of 
arsenious  oxide  and  heat 
on  platinum  foil. 
Unpleasant  smell  of  cacodyl 
=  acetic  acid. 

(d)  Add  a  few  drops  of  mercuric 

chloride  solution  in  test- 
tube,  and  heat  to  70°  C. 
Precipitate  of  mercurous 
chloride  which  is  slowly 
reduced  to  mercury  = 
formic  acid. 


284  METHODS    OF   IDENTIFICATION  AND    STUDY 

8.  If  the  distillation  of  "  B"  is  continued  as  long  as 
acid  conies  over    (distilled  water  being  occasionally 
added  to   the  distilling  flask)    the   distillate   can   be 
measured  and  50   c.c.   used  for  tit  ration.     This  will 
give  the  amount  of  volatile  acid  formation. 

9.  The  second  part  of  the  filtrate  "B"   (see  page 
282)    should  be  examined  for  lactic,  oxalic,  succinic, 
benzoic,  salicylic,  gallic  and  tannic  acids,  as  follows : 

Ether  Soluble  Acids. — 

1.  Evaporate  to  a  thin  syrup,  acidify  strongly  with 
phosphoric  acid. 

2.  Extract  with  five  times  its  volume  of  ether  by 
agitation  in  a  separatory  funnel. 

3.  Evaporate  the  ethereal  extract  to  a  thin  syrup. 

4.  Add  100  c.c.  water  and  mix  thoroughly. 

5.  To  a  small  portion  of  this  solution  add  slight  ex- 
cess of  sodium  carbonate,  evaporate  to  dryness  on  the 
water-bath,  dissolve  in  5-10  c.c.  pure  sulphuric  acid,  add 
2  drops  saturated  copper  sulphate  solution,  place  in  a 
test-tube  and  heat  in  a  boiling  water- bath  for  2  minutes, 
cool,  add  2  or  3  drops  of  the  alcoholic  thiophene  and 
warm  gently. 

Cherry  red  colour  =  lactic  acid. 

If  a  brown  colour  is  produced  on  the  addition  of 
sulphuric  acid,  another  sample  should  be  taken  and 
boiled  with  animal  charcoal  before  evaporating. 

6.  If  lactic  acid  is  definitely  present,  prepare  zinc 
lactate  by  boiling  part  of  the  solution  of  the  ether  extract 
with  excess  of  zinc  carbonate,  filtering  and  evaporating 
to  crystallise.     The  crystals  so  obtained  have  a  char- 
acteristic form,  and  if  dried  at  110°  C.,  should  contain 
26.87  per  cent,  of  zinc. 

7 .  Test  a  portion  of  the  rest  of  the  solution  of  the  ether 
extract  for  oxalic  acid  (page  282,  step  5).     Carefully 
neutralise  the  remainder  and  add  ferric  chloride  solution. 

Red    brown    gelatinous    precipitate  =  succinic    acid. 


ALCOHOL    PRODUCTION  285 

Buff  precipitate  =  benzole  acid,  and  other  acids  re- 
lated to  benzoic  acid. 

Violet  colour  =  salicylic  acid. 

Inky  black  colour  or  precipitate = gallic  acid  or  tannic 
acid. 

For  further  identification  the  melting-points  of  the 
crystalline  acids,  and  the  percentage  of  silver  in  their 
silver  salts  should  be  determined. 

3.  Ammonia  Production. — 

Medium  Required: 

Nutrient  bouillon. 
Reagent  Required: 

Nessler  reagent. 

METHOD. — 

1.  Prepare  cultivation  in  bulk  (100  c.c.)  in  a  250  c.c. 
flask  and  incubate  together  with  a  control  flask. 

Test  the  cultivation  and  the  control  for  ammonia 
in  the  following  manner : 

2.  To  each  flask  add  2  grammes  of  calcined  mag- 
nesia, then  connect  up  with  condensers  and  distil. 

3 .  Collect  50  c.c.  distillate,  from  each,  in  a  Nessler  glass. 

4.  Add  i  c.c.  Nessler  reagent  to  each  glass  by  means 
of  a  clean  pipette. 

Yellow  colour  =  ammonia. 

The  depth  of  colour  is  proportionate  to  the  amount 
present. 

4.  Alcohol,  etc.,  Production. — Divide  the  distillate 
"A"  obtained  in  the  course  of  a  previous  experiment 
(vide  page  282,  step  3)  into  four  portions  and  test  for 
the  production  of  alcohol,  acetaldehyde,  acetone. 

i.  Add  Lugol's  iodine,  then  a  little  NaOH  solution, 
and  stir  with  a  glass  rod  till  the  colour  of  the  iodine 
disappears. 

Pale-yellow  crystalline  precipitate  of  iodoform,  with 
its  characteristic  smell,  appearing  in  the  cold,  indicates 
acetaldehyde,  or  acetone ;  appearing  only  on  warming 
indicates  alcohol. 


286  METHODS    OF   IDENTIFICATION   AND    STUDY 

The  precipitate  may  be  absent  even  when  the  odour 
is  pronounced. 

2.  Add  SchifFs  reagent. 

Violet  or  red  colour  =  aldehyde. 

3.  To  10  c.c.  of  solution  add  2.5  c.c.,  25  per  cent, 
sulphuric  acid,    and  a  crystal  or  two   of  potassium 
bichromate  and  distil.     Reduction  of  the  bichromate 
to  a  green  colour  and  a  distillate,  which  smells  of  acet- 
aldehyde  and  reacts  with  Schiff's  reagent,  shows  the 
presence  of  alcohol  in  the  original  liquid. 

4.  Add  a  few  drops  of  sodium  nitroprusside  solution, 
make    alkaline    with    ammonia,    then    saturate    with 
ammonium    sulphate    crystals.     Acetone    gives    little 
colour  on  the  addition  of  ammonia,  but  after  the  addi- 
tion  of   ammonium   sulphate   a   deep   permanganate 
colour,  which  takes  ten  minutes  to  reach  its  full  inten- 
sity.    Aldehyde   gives   a   carmine   red   unaltered   by 
ammonium  sulphate. 

5.  Indol  Production. — 

Media  Required: 

Inosite-free  bouillon  (vide  page  183). 
Or  peptone  water  (vide  page  177). 
Reagents  Required: 

Potassium  persulphate,  saturated  aqueous  solution. 
Paradimethylamino-benzaldehyde  solution.     This  is  prepared  by 
mixing: 

Paradimethylamino-benzaldehyde     ...        4  grammes 

Absolute  alcohol 380  c.c. 

Hydrochloric  acid,  concentrated   ....     80  c.c. 

METHOD. — 

Prepare  several  test-tube  cultivations  of  the 
organism  to  be  tested,  and  incubate. 

Test  for  indol  by  means  of  the  Rosindol  reaction  in 
the  following  manner.  (If  the  culture  has  been  incu- 
bated at  37°  C.,  it  must  be  allowed  to  cool  to  the  room 
temperature  before  applying  the  test.) 

i.  Remove  2  c.c.  of  the  cultivation  by  means  of  a 
sterile  pipette  and  transfer  to  a  clean  tube,  then, 


PHENOL    PRODUCTION  287 

2.  Add    2     c.c.     paradimethylamino-benzaldehyde 
solution. 

3.  Add  2  c.c.  potassium  persulphate  solution. 

The  presence  of  indol  is  indicated  by  the  appearance 
of  a  delicate  rose-pink  colour  throughout  the  mixture 
which  deepens  slightly  on  standing. 

Indol  is  tested  for  in  many  laboratories  by  the  ordinary 
nitrosoindol  reaction  which,  however,  is  not  so  delicate  a  method 
as  that  above  described.  The  test  is  carried  out  as  follows: 

1.  Remove  the  cotton- wool  plug  from  the  tube,  and  run  in 
i  c.c.  pure  concentrated  sulphuric  acid  down  the  side  of  the  tube 
by  means  of  a  sterile  pipette.     Place  the  tube  upright  in  a  rack, 
and  allow  it  to  stand,  if  necessary,  for  ten  minutes. 

A  rose-pink  or  red  colour  at  the  junction  of  the  two  liquids  = 
indol  (plus  a  nitrite}. 

2.  If  the  colour  of  the  medium  remains  unaltered,  add  2  c.c.  of 
a  o.o i  per  cent,  aqueous  solution  sodium  nitrite,  and  again  allow 
the  culture  to  stand  for  ten  minutes. 

Red  colouration  =  indol. 

NOTE. — In  place  of  performing  the  test  in  two  stages  as  given 
above,  2  c.c.  concentrated  commercial  sulphuric,  hydrochloric,  or 
nitric  acid  (all  of  which  hold  a  trace  of  nitrite  in  solution) ,  may  be 
run  into  the  cultivation.  The  development  of  a  red  colour  within 
twenty  minutes  will  indicate  the  presence  of  indol. 

5a.  Phenol  Production. — 

Medium  Required: 

Nutrient  bouillon. 
Reagents  Required: 

Hydrochloric  acid,  concentrated. 

Millon's  reagent. 

Ferric  chloride,  i  per  cent,  aqueous  solution. 

METHOD. — 

1.  Prepare  cultivation  in  a  Bohemian  flask  contain- 
ing at  least  50  c.c.  of  medium,  and  incubate. 

Test  for  phenol  in  the  following  manner : 

2.  Add   5   c.c.,  25   per  cent,  sulphuric  acid  to  the 
cultivation  and  connect  up  the  flask  with  a  condenser. 

3.  Distil  over   15  to   20  c.c.     Divide  the  distillate 
into  three  portions  a,  b  and  c. 

4.  Add  to  (a)  0.5  c.c.  Millon's  reagent  and  boil. 
Red  colour  =  phenol. 


288  METHODS    OF   IDENTIFICATION    AND    STUDY 

5.  Add  to  (b)  about  0.5  c.c.  ferric  chloride  solution. 
Violet  colour  =  phenol. 

(If  the  distillate  be  acid  the  reaction  will  be  nega- 
tive.) 

6.  Add  to  (c)  bromine  water.     Crystalline  white  ppt. 
of  tribromo-phenol  =  phenol. 

NOTE. — If  both  indol  and  phenol  appear  to  be  present  in  culti- 
vations of  the  same  organism,  it  is  well  to  separate  them  before 
testing.  This  may  be  done  in  the  following  manner: 

1.  Prepare  inosite-free  bouillon  cultivation,  say  200 
or  300  c.c.,  in  a  flask  as  before. 

2.  Render  definitely  acid  by  the  addition  of  acetic 
acid  and  connect  up  the  flask  with  a  condenser. 

3.  Distil  over  50  to  70  c.c. 

Distillate  will  contain  both  indol  and  phenol. 

4.  Render    the    distillate    strongly    alkaline    with 
caustic  potash  and  redistil. 

Distillate  will  contain  indol;  residue  will  contain 
phenol. 

5.  Test  the  distillate  for  indol  (vide  ante). 

6.  Saturate   the   residue,    when   cold,    with   carbon 
dioxide  and  redistil. 

7.  Test  this  distillate  for  phenol  (vide  ante). 

6.  Pigment  Production. — 

1.  Prepare  tube  cultivations  upon  the  various  media 
and  incubate  under  varying  conditions  as  to  tempera- 
ture (at  37°  C.  and  at  20°  C.),  atmosphere  (aerobic  and 
anaerobic) ,  and  light  (exposure  to  and  protection  from) . 

Note  the  conditions  most  favorable  to  pigment 
formation. 

2.  Note  the  solubility  of  the  pigment  in  various 
solvents,  such  as  water  (hot  and  cold),  alcohol,  ether, 
chloroform,  benzol,  carbon  bisulphide. 

3.  Note  the  effect  of  acids  and  alkalies  respectively 
upon  the  pigmented  cultivation,  or  upon  solutions  of 
the  pigment. 


GAS    PRODUCTION  289 

4.  Note  spectroscopic  reactions. 

7.  Reducing  Agent  Formation. — 

(a)  Colour  Destruction. — 

1 .  Prepare  tube  cultivations  in  nutrient  bouillon  tin- 
ted with  litmus,  rosolic  acid,  neutral  red,  and  incubate. 

2.  Examine  the  cultures  each  day  and  note  whether 
any  colour  change  occurs. 

(6)  Nitrates  to  Nitrites. — 

Medium  Required: 

Nitrate  bouillon  (vide  page  185). 

Or  nitrate  peptone  solution  (vide  page  186). 
Reagents  Required: 

Sulphuric  acid  (25  per  cent.). 

Metaphenylene  diamine,  5  per  cent,  aqueous  solution. 

METHOD. — 

1.  Prepare  tube  cultivations  and  incubate  together 
with  control  tubes   (i.  e.,  uninoculated  tubes  of  the 
same  medium,  placed  under  identical  conditions  as  to 
environment) . 

This  precaution  is  necessary  as  the  medium  is  liable 
to  take  up  nitrites  from  the  atmosphere,  and  an 
opinion  as  to  the  absence  of  nitrites  in  the  cultivation 
is  often  based  upon  an  equal  colouration  of  the  medium 
in  the  control  tube. 

Test  both  the  culture  tube  and  the  control  tube  for 
the  presence  of  nitrites. 

2.  Add  a  few  drops  of  sulphuric  acid  to  the  medium 
in  each  of  the  tubes. 

3.  Then  run  in  2  or  3  c.c.  metaphenylene  diamine 
into  each  tube. 

Brownish-red  colour  =  nitrites. 
The  depth  of  colour  is  proportionate  to  the  amount 
present. 

8.  Gas  Production. — 

(A)  Carbon  Dioxide  and  Hydrogen. — 

Apparatus  Required: 

Fermentation  tubes  (vide  page   161)   containing  sugar  bouillon 
19 


METHODS    OF   IDENTIFICATION   AND    STUDY 

(glucose,  lactose,  etc.).     The  medium  should  be  prepared  from 
inosite-free  bouillon  (vide  page  183). 
Reagent  Required: 

-  caustic  soda. 
2 

METHOD. — 

1.  Inoculate  the  surface  of  the  medium  in  the  bulb 
of  a  fermentation  tube  and  incubate. 

2.  Mark  the  level  of  the  fluid  in  the  closed  branch  of 
the  fermentation  tube,  at  intervals  of  twenty-four  hours, 
and  when  the  evolution  of  gas  has  ceased,  measure  the 
length  of  the  column  of  gas  with  the  millimetre  scale. 

Express  this  column  of  gas  as  a  percentage  of  the 
entire  length  of  the  closed  branch. 

3.  To  analyse  the  gas  and  to  determine  roughly  the 
relative  proportions  of  C02  and  H2,  proceed  as  follows : 

Fill  the  bulb  of  the  fermentation  tube  with  caustic 
soda  solution. 

Close  the  mouth  of  the  bulb  with  a  rubber  stopper. 

Alternately  invert  and  revert  the  tube  six  or  eight 
times,  to  bring  the  soda  solution  into  intimate  contact 
with  the  gas. 

Return  the  residual  gas  to  the  end  of  the  closed 
branch,  and  measure. 

The  loss  in  volume  of  gas  =  carbon  dioxide. 

The  residual  gas  =  hydrogen. 

Transfer  gas  to  the  bulb  of  the  tube,  and  explode  it 
by  applying  a  lighted  taper. 

(B)  Sulphuretted  Hydrogen. — 

Media  Required: 

Iron  peptone  solution  (vide  page  185). 
Lead  peptone  solution. 

1.  Inoculate  tubes  of  media,  and  incubate  together 
with  control  tubes. 

2.  Examine  from  day  to  day,  at  intervals  of  twenty- 
four  hours. 

The  liberation  of  the  H2S  will  cause  the  yellowish- 


GAS    PRODUCTION 


291 


white  precipitate  to  darken  to  a  brownish- black,  or 
jet  black,  the  depth  of  the  colour  being  proportionate 
to  the  amount  of  sulphuretted  hydrogen  present. 

Quantitative :  For  exact  quantitative  analyses  of  the 
gases  produced  by  bacteria  from  certain  media  of 
definite  composition,  the  methods  devised  by  Pakes 
must  be  employed,  as  follows : 

Apparatus  Required: 

Bohemian  flask  (300  to  1500  c.c.  capacity)  containing  from 
100  to  400  c.c.  of  the  medium.  The  mouth  of  the  flask  is  fitted 


FIG.  153. — Gas-collecting  apparatus. 

with  a  perforated  rubber  stopper,  carrying  an  L-shaped  piece  of 
glass  tubing  (the  short  arm  passing  just  through  the  stopper). 
To  the  long  arm  of  the  tube  is  attached  a  piece  of  pressure  tubing 
some  8  cm.  in  length,  plugged  at  its  free  end  with  a  piece  of 
cotton-wool.  Measure  accurately  the  total  capacity  of  the  flask 
and  exit  tube,  also  the  amount  of  medium  contained.  Note  the 
difference. 

Gas  receiver.  This  is  a  bell  jar  of  stout  glass,  14  cm.  high  and 
9  cm.  in  diameter.  At  its  apex  a  glass  tube  is  fused  in.  This 
rises  vertically  5  cm.,  and  is  then  bent  at  right  angles,  the  horizon- 
tal arm  being  10  cm.  in  length.  A  three-way  tap  is  let  hori- 
zontally into  the  vertical  tube  just  above  its  junction  with  the 
bell  jar. 

An  iron  cylinder  just  large  enough  to  contain  the  bell  jar. 

About  1 5  kilos  of  metallic  mercury. 

Melted  paraffin. 


2Q2  METHODS    OF   IDENTIFICATION  AND    STUDY 

An  Orsat-Lunge  working  with  mercury  instead  of  water, 
provided  with  two  gas  tubes  of  extra  length  (capacity  120  and 
60  c.c.  respectively  and  graduated  throughout,  both  being  water- 
jacketed)  or  other  gas  analysis  apparatus,  capable  of  dealing  with 
CO2,  O2,  H2,  and  N2. 

METHOD.— 

i.  Inoculate  the  medium  in  the  flask  in  the  usual 
manner,  by  means  of  a  platinum  needle,  taking  care 
that  the  neck  of  the  flask  and  the  rubber  stopper  are 


FIG.  154. — Orsat-Lunge  gas  analysis  apparatus. 

thoroughly   flamed    before   and   after   the  operation. 

2.  Fill  the  iron  cylinder  with  mercury. 

3.  Place   the    bell    jar   mouth    downward    in    the 
mercury — first  seeing  that  there  is  free  communication 
between  the  interior  of  the  jar  and  the  external  air — and 
suck  up  the  mercury  into  the  tap ;  then  shut  off  the  tap. 

4.  Plug  the  open  end  of  the  three-way  tap  with 
melted  wax. 

5.  Connect  up  the  horizontal  arm  of  the  culture 
flask  with  that  of  the  gas  receiver  by  means  of  the 


GAS    PRODUCTION  293 

pressure  tubing  (after  removing  the  cotton-wool  plug 
from  the  rubber  tube),  as  shown  in  Fig.  153. 

6.  Give  the  three-way  tap  half  turn  to  open  com- 
munication between  flask  and  receiver,  and  seal  all 
joints  by  coating  with  a  film  of  melted  wax.     When 
the  tap  is  turned,  the  mercury  in  the  receiver  will 
naturally  fall. 

7.  Place    the    entire    apparatus    in    the    incubator. 
(Two  hours  later,  by  which  time  the  temperature  of 
the   apparatus   is   that   of   the   incubator,    mark   the 
height  of  the  mercury  on  the  receiver.) 

8.  Examine  the  apparatus  from  day  to  day  and  mark 
the  level  of  the  mercury  in  the  receiver  at  intervals 
of  twenty-four  hours. 

9.  When  the  evolution  of  gas  has  ceased,  remove 
the  apparatus  from  the  incubator;  clear  out  the  wax 
from  the  nozzle  of  the  three-way  tap  (first  adjusting 
the  tap  so  that  no  escape  of  gas  shall  take  place)  and 
connect  it  with  the  Orsat. 

10.  Remove,  say,  100  c.c.  of  gas  from  the  receiver, 
reverse  the  tap  and  force  it  into  the  culture  flask. 
Remove    100   c.c.    of  mixed   gases   from  the   culture 
flask  and  replace  in  the  receiver. 

Repeat  these  processes  three  or  four  times  to  ensure 
thorough  admixture  of  the  contents  of  flask  and 
receiver. 

1 1 .  Now  withdraw  a  sample  of  the  mixed  gases  into 
the  Orsat  and  analyse. 

In  calculating  the  results  be  careful  to  allow  for  the 
volume  of  air  contained  in  the  flask  at  the  commence- 
ment of  the  experiment. 

For  the  collection  of  gases  formed  under  anaerobic 
conditions  a  slightly  different  procedure  is  adopted: 

1.  Fix  a  culture  flask  (500  c.c.  capacity)  with  a  per- 
forated rubber  stopper  carrying  an  L-shaped  piece  of 
manometer  tubing,  each  arm  5  cm.  in  length. 

2.  Prepare  a  second  L-shaped  piece  of  tubing,  the 


2Q4  METHODS    OF   IDENTIFICATION   AND    STUDY 

short  arm  5  cm.  and  the  long  arm  20  cm.,  and  connect 
its  short  arm  to  the  horizontal  arm  of  the  tube  in  the 
culture  flask  by  means  of  a  length  of  pressure  tubing, 
provided  with  a  screw  clamp. 

3.  Fill   the    culture   flask   completely   with    boiling 
medium  and  pass  the  long  piece  of  tubing  through  the 
plug  of  an  Erlenmeyer  flask  (150  c.c.  capacity)  which 
contains  100  c.c.  of  the  same  medium. 

4.  Sterilise  these  coupled  flasks  by  the  discontinuous 
method,  in  the  usual  manner. 

Immediately  the  last  sterilisation  is  completed, 
screw  up  the  clamp  on  the  pressure  tubing  which  con- 
nects them,  and  allow  them  to  cool. 

As  the  fluid  cools  and  contracts  it  leaves  a  vacuum 
in  the  neck  of  the  flask  below  the  rubber  stopper. 

5.  To  inoculate  the  culture  flask,  withdraw  the  long 
arm  of  the  bent  tube  from  the  Erlenmeyer  flask  and 
pass  it  to  the  bottom  of  a  test-tube  containing  a  young 
cultivation  (in  a  fluid  medium  similar  to  that  contained 
in  the  culture  flask)  of  the  organism  it  is  desired  to 
investigate. 

6.  Slightly  release  the  clamp  on  the  pressure  tub- 
ing to  allow  4  or  5  c.c.  of  the  culture  to  enter  the 
flask. 

7.  Clamp  the  rubber  tube  tightly;  remove  the  bent 
glass  tube  from  the  culture  tube  and  plunge  it  into  a 
flask  containing  recently  boiled  and  quickly  cooled  dis- 
tilled water. 

8.  Release  the  clamp  again  and  wash  in  the  remains 
of  the  cultivation  until  the  culture  flask  and  tubing 
are  completely  filled  with  water. 

9.  Clamp  the  rubber  tubing  tightly  and  take  away 
the  long-armed  glass  tubing. 

10.  Prepare   the   gas   receiver   as   in    the   previous 
method  (in  this  case,  however,  the  mercury  should  be 
warmed  slightly)  and  fill  the  horizontal  arm  of  the  re- 
ceiver with  hot  water. 


ATMOSPHERE  295 

11.  Connect  up  the  culture  flask  with  the  horizontal 
arm  of  the  gas  receiver. 

12.  Remove  the  screw  clamp  from  the  rubber  tubing, 
adjust  the  three-way  tap,  seal  all  joints  with  melted 
wax,  and  incubate. 

13.  Complete  the  investigation  as  described  for  the 
previous  method. 

BY  PHYSICAL  METHODS. 

Examine  cultivations  of  the  organism  with  reference 
to  its  growth  and  development  under  the  following 
headings : 
Atmosphere : 

(a)  In  the  presence  of  oxygen. 

(b)  In  the  absence  of  oxygen. 

(c)  In  the  presence  of  gases  other  than  oxygen. 
Temperature : 

(a)  Range. 

(b)  Optimum. 

(c)  Thermal  death-point: 
Moist:  Vegetative  forms. 

Spores. 
Dry:  Vegetative  forms. 

Spores. 

Reaction  of  medium. 
Resistance  to  lethal  agents: 

(a)  Desiccation. 

(b)  Light:  Diffuse. 

Direct. 
Primary  colours. 

(c)  Heat. 

(d)  Chemical  antiseptics  and  disinfectants. 
Vitality  in  artificial  cultures. 

I.  Atmosphere. — The  question  as  to  whether  the 
organism  under  observation  is  (a)  an  obligate  aerobe, 
(b)  a  facultative  anaerobe,  or  (c)  an  obligate  anaerobe 
is  roughly  decided  by  the  appearance  of  cultivations 


296  METHODS    OF   IDENTIFICATION  AND    STUDY 

in  the  fermentation  tubes.  Obvious  growth  in  the 
closed  branch  as  well  as  in  the  bulb  or  in  the  inverted 
gas  tube  as  well  as  in  the  bulk  of  the  medium  will 
indicate  that  it  is  a  facultative  anaerobe ;  whilst  growth 
only  occurring  in  the  bulb  or  in  the  closed  branch 
shows  that  it  is  an  obligate  aerobe  or  anaerobe  respec- 
tively. This  method,  however,  is  not  sufficiently 
accurate  for  the  present  purpose,  and  the  examination 
of  an  organism  with  respect  to  its  behaviour  in  the 
absence  of  oxygen  is  carried  out  as  follows : 

Apparatus  Required: 

Buchner's  tubes. 

Bulloch's  apparatus. 

Exhaust  pump. 

Pyrogallic  acid. 

Dekanormal  caustic  soda. 
Media  Required: 

Glucose  formate  agar. 

Glucose  formate  gelatine. 

Glucose  formate  bouillon. 

METHOD. — 

i .  Prepare  four  sets  of  cultivations : 

(A)  Sloped    glucose    formate    agar,    and    incubate 
aerobically  at  37°  C. 

Sloped  glucose  formate  gelatine,  and  incubate  aero- 
bically at  20°  C. 

(B)  Sloped  glucose  agar  to  incubate  anaerobically 
at37°C. 

Sloped    glucose    formate   gelatine    to    incubate 
anaerobically  at  20°  C. 

(C)  Sloped  glucose  formate  agar  to  incubate  anaero- 
bically at  37°  C. 

Glucose  formate  bouillon  to  incubate  anaerobi- 
cally at  37°  C. 

(D)  Sloped   glucose   formate  gelatine   to   incubate 
anaerobically  at  20°  C. 

Glucose  formate  bouillon  to  incubate  anaerobi- 
cally  at  20°  C. 


ATMOSPHERE  297 

2.  Seal  the  cultures  forming  set   B   in    Buchner's 
tubes  (vide  page  239). 

3 .  Seal  the  cultures  forming  set  C  in  Bulloch's  appara- 
tus; exhaust  the  air  by  means  of  a  vacuum  pump,  and 
provide  for  the  absorption  of  any  residual  oxygen  by  the 
introduction  of  pyrogallic  acid  and  caustic  soda  in  solu- 
tion (vide  page  245).     Treat  set  D  in  the  same  way. 

4.  Observe    the    cultivations    macroscopically    and 
microscopically  at  intervals  of  twenty-four  hours  until 
the  completion,  if  necessary,  of  seven  days'  incubation. 

5.  Control  these  results. 
Gases  Other  than  Oxygen. — 

Apparatus  Required: 

Bulloch's  apparatus. 

Sterile  gas  filter  (vide  page  40). 

Gasometer  containing  the  gas  it  is  desired  to  test  (SO2,  N2O, 
NO,  CO2,    etc.)  or  gas  generator  for  its  production. 

METHOD. — 

1.  Prepare  at   least  seven  tube  cultivations  upon 
solid  media  and  deposit  them  in  Bulloch's  apparatus. 

2.  Connect  up  the  inlet  tube  of  the  Bulloch's  jar  with 
the  sterile  gas  filter,  and  this  again  with  the  delivery 
tube  of  the  gasometer  or  gas  generator. 

3.  Open  both  stop-cocks  of  the  Bulloch's  apparatus 
and  pass  the  gas  through  until  it  has  completely  re- 
placed the  air  in  the  bell  jar  as  shown  by  the  result  of 
analyses  of  samples  collected  from  the  exit  tube. 

4.  Incubate  under  optimum  conditions  as  to  tem- 
perature. 

5.  Examine  the  cultivations  at  intervals  of  twenty- 
four  hours,  until  the  completion  of  seven  days. 

6.  Remove  one  tube  from  the  interior  of  the  appara- 
tus each  day.     If  no  growth  is  visible,  incubate  the 
tube  under  optimum  conditions  as  to  temperature  and 
atmosphere,  and  in  this  way  determine  the  length  of 
exposure  to  the  action  of  the  gas  necessary  to  kill  the 
organisms  under  observation. 

7.  Control  these  results. 


298  METHODS    OF   IDENTIFICATION   AND    STUDY 

II.  Temperature.— 

(A)  Range. — 

1.  Prepare  a  series  of  ten  tube  cultivations,  in  fluid 
media,  of  optimum  reaction. 

2.  Arrange  a  series  of  incubators  at  fixed  tempera- 
tures, varying  5°  C.  and  including  temperatures  between 
5°  C.  and  50°  C. 

(In  the  absence  of  a  sufficient  number  of  incubators 
utilise  the  water-bath  employed  in  testing  the  thermal 
death-point  of  vegetative  forms.) 

3.  Incubate  one  tube  cultivation  of  the  organism 
aerobically  or  anaerobically,  as  may  be  necessary,  in 
each  incubator,  and  examine  at  half -hour  intervals  for 
from  five  to  eighteen  hours. 

4.  Note  that  temperature  at  which  growth  is  first 
observed  macroscopically  (Optimum  temperature). 

5.  Continue  the  incubation  until  the  completion  of 
seven  days.     Note  the  extremes  of  temperature  at 
which  growth  takes   place    (Range  of  temperature). 

6.  Control    these   results — if    considered    necessary 
arranging  the  series  of  incubators  to  include  each  degree 
centigrade  for  five  degrees  beyond  each  of  the  extremes 
previously  noted. 

(B)  Optimum. — 

1.  Prepare  a  second  series  of  ten  tube  cultivations 
under  similar  conditions  as  to  reaction  of  medium. 

2.  Incubate  in  a  series  of  incubators  in  which  the 
temperature  is  regulated  at  intervals  of  i°  C.  for  five 
degrees  on  either  side  of  optimum  temperature  observed 
in  the  previous  experiment  (A,  step  4). 

3 .  Observe  again  at  half -hour  intervals  and  note  that 
temperature  at  which  growth  is  first  visible  to  the 
naked  eye  =  Optimum  temperature. 

(Q   Thermal  Death-point  (t.  d.  p.) — 

Moist — Vegetative  Forms : 

The  /.  d.  p.  here  is  that  temperature  which  with 


TEMPERATURE 


299 


certainty  kills  a  watery  suspension  of  the  organisms  in 
question  after  an  exposure  of  10  minutes. 

Apparatus  Required: 

Water-bath.  For  the  purpose  of  observing  the  thermal  death- 
point  a  special  water-bath  is  necessary.  The  temperature  of  this 
piece  of  apparatus  is  controlled  by  means  of  a  capsule  regulator 


FIG.  155. — Hearson's  water-bath. 

that  can  be  adjusted  for  intervals  of  half  a  degree  centigrade 
through  a  range  of  30°,  from  50°  C.  to  80°  C.  by  means  of  a  spring, 
actuated  by  the  handle  a,  which  increases  the  pressure  in  the 
interior  of  the  capsule.  A  hole  is  provided  for  the  reception  of 
the  nozzle  of  a  blast  pump,  so  that  a  current  of  air  may  be  blown 
through  the  water  while  the  bath  is  in  use,  and  thus  ensure  a 
uniform  temperature  of  its  contents.  Through  a  second  hole  is 
suspended  a  certified  centigrade  thermometer,  the  bulb  of  which 
although  completely  immersed  in  the  water  is  raised  at  least  2  cm. 
above  the  floor  of  the  bath. 

Sterile  glass  capsules. 

Flask  containing  250  c.c.  sterile  normal  saline  solution. 

Case  of  sterile  pipettes,  10  c.c.  (in  tenths  of  a  cubic  centimetre). 

Special  platinum  loop. 

Test-tubes,  18  by  1.5  cm.,  of  thin  German  glass. 

Case  of  sterile  petri  dishes. 

Tubes  of  agar  or  gelatine. 


300          METHODS  OF  IDENTIFICATION  AND  STUDY 

METHOD.-— 

1.  Prepare  tube  cultivations  on  solid  media  of  opti- 
mum reaction ;  incubate  forty-eight  hours  under  opti- 
mum conditions  as  to  temperature  and  atmosphere. 

2 .  Examine  preparations  from  the  cultivation  micro- 
scopically to  determine  the  absence  of  spores. 

3.  Pipette  5  c.c.  salt  solution  into  each  of  twelve 
capsules. 

4.  Suspend   three   loopfuls   of    the   surface  growth 
(using  a  special  platinum  loop,  vide  page  316)  in  the 
normal  saline  solution  by  emulcifying  evenly  against 
the  moist  walls  of  each  capsule. 

5.  Transfer  emulsion  from  each  capsule  to  sterile 
250  c.c.  flask,  and  mix. 

6.  Pipette  5  c.c.  emulsion  into  each  of  twelve  sterile 
test-tubes  numbered  consecutively. 

7.  Adjust  the  first  tube  in  the  water-bath,  regulated 
at  40°  C.,  by  means  of  two  rubber  rings  around  the 
tube,  one  above  and  the  other  below  the  perforated 
top  of  the  bath,  so  that  the  upper  level  of  the  fluid 
in  the  tube  is  about  4  cm.  below  the  surface  of  the  water 
in  the  bath,  and  the  bottom  of  the  tube  is  a  similar 
distance  above  the  bottom  of  the  bath. 

8.  Arrange  a  control  test-tube  containing  5  c.c.  sterile 
saline    solution    under   similar  conditions.     Plug  the 
tube  with  cotton -wool  and  pass  a  thermometer  through 
the  plug  so  that  its  bulb  is  immersed  in  the  water. 

9.  Close  the  unoccupied  perforations  in  the  lid  of 
the  water-bath  by  means  of  glass  balls. 

10.  Watch  the  thermometer  in  the  test-tube  until  it 
records  a  temperature  of  40°  C.     Note  the  time.     Ten 
minutes  later  remove  the  tube  containing  the  sus- 
pension, and  cool  rapidly  by  immersing  its  lower  end  in 
a  stream  of  running  water. 

11.  Pour  three  gelatine  (or  agar)  plates  containing 
respectively  0.2,  0.3,  and  0.5  c.c.  of  the  suspension, 
and  incubate. 


TEMPERATURE  301 

12.  Pipette  the  remaining  4  c.c.  of  the  suspension 
into  a  culture  flask  containing  250  c.c.   of  nutrient 
bouillon,  and  incubate. 

13.  Observe  these  cultivations  from  day  to  day. 
"  No  growth"  must  not  be  recorded  as  final  until  after 
the  completion  of  seven  days'  incubation. 

14.  Extend   these   observations   to    the   remaining 
tubes  of  the  series,  but  varying  the  conditions  so  that 
each  tube  is  exposed  to  a  temperature  2°  C.  higher 
than  the  immediately  preceding  one — i.  e.,  42°  C.,  44° 
C.,  46°  C.,  and  so  on. 

15.  Note  that  temperature,  after  exposure  to  which 
no  growth  takes  place  up  to  the  end  of  seven  days* 
incubation,  =  the  thermal  death-point. 

1 6.  If  greater  accuracy  is  desired,  a  second  series 
of  tubes  may  be  prepared  and  exposed  for  ten  minutes 
to  fixed  temperatures  varying  only  0.5°  C.,  through 
a  range  of  5°  C.  on  either  side  of  the  previously  observed 
death-point. 

Moist — Spores :  The  thermal  death-point  in  the  case 
of  spores  is  that  time  exposure  to  a  fixed  temperature  of 
100°  C.  necessary  to  effect  the  death  of  all  the  spores 
present  in  a  suspension. 

NOTE. — If  it  is  desired  to  retain  the  time  constant  10  minutes 

and  investigate  the  temperature  necessary  to  destroy  the  spores, 
varying  amounts  of  calcium  chloride  must  be  added  to  the  water 
in  the  bath,  when  the  boiling-point  will  be  raised  above  100°  C. 
according  to  the  percentage  of  calcium  in  solution.  In  such  case 
use  the  bath  figured  on  page  227  the  bath  figured  on  page  299  can 
only  be  used  if  the  capsule;  is  first  removed. 

It  is  determined  in  the  following  manner 

Apparatus  Required: 

Steam-can  fitted  with  a  delivery  tube  and  a  large  bore  safety- 
valve  tube. 

Water-bath  at  100°  C. 

Erlenmeyer  flask,  500  c.c.  capacity,  containing  140  c.c.  sterile 
normal  saline  solution  and  fitted  with  rubber  stopper  perforated 
with  four  holes. 

The  rubber  stopper  is  fitted  as  follows: 


METHODS    OF   IDENTIFICATION  AND    STUDY 


(a) 


Thermometer  to  120°  C.,  its  bulb  immersed  in  the  normal 
saline. 

Straight  entry  tube,  reaching  to  the  bottom  of  the  flask, 
the  upper  end  plugged  with  cotton-wool. 
Bent  syphon  tube,  with  pipette  nozzle  attached  by  means 
of  rubber  tubing  and  fitted  with  pinch-cock. 
The   nozzle   is   protected    from    accidental    contamination    by 
passing  it  through  the  cotton-wool  plug  of  a  small  test-tube. 


(b) 

(c) 


FlG.  156. — Apparatus  arranged  for  the  determination  of  the  death -point  of, 

spores. 

(d)  A  sickle-shaped  piece  of  glass  tubing  passing  just  through 
the  stopper,  plugged  with  cotton-wool,  to  act  as  a  vent 
for  the  steam. 
Sterile  plates. 
Sterile  pipettes. 

Sterile  test-tubes  graduated  to  contain  5  c.c. 
Media  Required: 
Gelatine  or  agar. 
Culture  flasks  containing  200  c.c.  nutrient  bouillon. 

METHOD.— 

i.  Prepare  twelve  tube  cultivations  upon  the  sur- 
face '(or  two  cultures  in  large  flat  culture  bottles — 


TEMPERATURE 


303 


vide  page  5)  of  nutrient  agar  and  incubate  under  the 
optimum  conditions  (previously  determined),  for  the 
formation  of  spores. 

Examine  preparations  from  the  cultures  micro- 
scopically to  determine  the  presence  of  spores. 

2.  Pipette  5  c.c.  sterile  normal  saline  into  each  cul- 
ture tube  or  30  c.c.  into  each  bottle  and  by -means  of  a 
sterile  platinum  spatula  emulsify  the  entire  surface 
growth  with  the  solution. 

3.  Add  the  60  c.c.  emulsion  to  140  c.c.  normal  saline 
contained  in  the  fitted  Erlenmeyer  flask. 

4.  Place  the  flask  in  the  water-bath  of  boiling  water. 

5.  Connect  up  the  straight  tube,  after  removing  the 
cotton-wool  plug,  with  the  delivery  tube  of  the  steam 
can ;  remove  the  plug  from  the  vent  tube. 

6.  When  the  thermometer  reaches  100°  C.,  open  the 
spring  clip  on  the  syphon,  discard  the  first  cubic  centi- 
meter of  suspension  that  syphons  over  (i.e.,  the  con- 
tents of  the  syphon  tube) ;  collect  the  next  5  c.c.  of 
the  suspension  in  the  sterile  graduated  test-tube  and 
pour  plates  and  prepare  flask  cultures  therefrom  as  in 
the  previous  experiments. 

7.  Repeat  this  process  at  intervals  of  twenty-five 
minutes'  steaming. 

8.  Observe  the  inoculated  plates  and  flasks  up  to 
the  completion,  if  necessary,  of  seven  days'  incubation. 

9.  Control  these  experiments,  but  in  this  instance 
syphon  off  portions  of  the  suspension  at  intervals  of 
one-half  to  one  minute  during  the  five  or  ten  minutes 
preceding  the  previously  determined  death-point. 

Thermal  Death-point. — 

Dry — Vegetative  Forms:  The  thermal  death-point 
in  this  case  is  that  temperature  which  with  certainty 
kills  a  thin  film  of  the  organism  in  question  after  a 
time  exposure  of  ten  minutes. 

Apparatus  Required: 

Hot-air  oven,  provided  with  thermo-regulator. 


304  METHODS    OF   IDENTIFICATION   AND    STUDY 

Sterile  cover-slips. 

Flask  containing  250  c.c.  sterile  normal  saline  solution. 

Case  of  sterile  pipettes,  10  c.c.  (in  tenths  of  a  cubic  centimetre). 

Case  of  sterile  capsules. 

Crucible  tongs. 

METHOD. — 

1.  Prepare  an  emulsion  with  three  loopfuls  from  an 
optimum  cultivation  in  5  c.c.  normal  saline  in  a  ster- 
ile capsule  and  examine  microscopically  to  determine 
the  absence  of  spore  forms. 

2.  Make  twelve  cover-slip  films  on  sterile  cover-slips; 
place  each  in  a  sterile  capsule  to  dry. 

3.  Expose  each  capsule  in  turn  in  the  hot-air  oven 
for  ten  minutes  to  a  different  fixed  temperature,  vary- 
ing 5°  C.  between  60°  C.  and  120°  C. 

4.  Remove  each  capsule  from  the  oven  with  crucible 
tongs  immediately  the  ten  minutes   are   completed; 
remove  the  cover-glass  from  its  interior  with  a  sterile 
pair  of  forceps. 

5.  Deposit  the  film  in  a  flask  containing  200  c.c. 
nutrient  bouillon. 

6.  Prepare  subcultivations  from  such  flasks  as  show 
evidence  of  growth,  to  determine  that  no  accidental 
contamination  has  taken  place  but  that  the  organism 
originally  spread  on  the  film  is  responsible  for  the 
growth. 

7.  Control  the  result  of  these  experiments. 

Dry — Spores :  The  thermal  death-point  in  this  case 
is  that  temperature  which  with  certainty  kills  the  spores 
of  the  organism  in  question  when  present  in  a  thin  film 
after  a  time  exposure  of  10  minutes. 

Apparatus  Required: 
As  for  vegetative  forms. 

METHOD. — 

i.  Prepare  a  sloped  agar  tube  cultivation  and  incu- 
bate under  optimum  conditions  as  to  spore  formations. 


REACTION    OF    MEDIUM  305 

2.  Pipette  5  c.c.  sterile  normal  saline  into  the  culture 
tube  and  emulsify  the  entire  surface  growth  in  it.     Ex- 
amine microscopically  to  determine  the  presence  of 
spores  in  large  numbers. 

3.  Spread  thin  even  films  on  twelve  sterile  cover- 
slips  and  place  each  cover-slip  in  a  separate  sterile 
capsule. 

4.  Expose  each  capsule  in  turn  for  ten  minutes  to  a 
different  fixed  temperature,   varying   5°  C.,   between 
100°  C.  and  160°  C. 

5.  Complete  the  examination  as  for  vegetative  forms. 

III.  Reaction  of  Medium. 

(A)  Range. — 

1.  Prepare  a  bouillon  culture  of  the  organism  and  in- 
cubate, under  optimum  conditions  as  to  temperature 
and  atmosphere,  for  twenty-four  hours. 

2.  Pipette  o.i   c.c.   of  the   cultivation  into  a  ster- 
ile  capsule;   add    9.9    c.c.    sterile    bouillon   and   mix 
thoroughly. 

3.  Prepare  a  series  of  tubes  of  nutrient  bouillon  of 
varying  reactions,  from  +25  to  —30  (vide  page  155), 
viz.:  +25,   +20,   +15,    +10,    +5,  neutral,    -5,    -10, 

-15.  -20>  ~25>  -3°- 

4.  Inoculate  each  of  the  bouillon  tubes  with  o.i  c.c. 
of  the  diluted  cultivation  by  means  of  a  sterile  gradu- 
ated pipette  and  incubate  under  optimum  conditions. 

5.  Observe    the    cultures    at    half -hourly    intervals 
from  the  third  to  the  twelfth  hours.     Note  the  reaction 
of  the  tube  or  tubes  in  which  growth  is  first  visible 
macroscopically  (probably  optimum  reaction) . 

6.  Continue  the  incubation  until  the  completion,  if 
necessary,  of  seven  days.     Note  the  extremes  of  acidity 
and   alkalinity   in    which   macroscopical   growth   has 
developed  (Range  of  reaction) . 

7.  Control  the  result  of  these  observations. 

(B)  Optimum  Reaction. — The  optimum  reaction  has 

20 


306  METHODS    OF   IDENTIFICATION   AND    STUDY 

already  been  roughly  determined  whilst  observing  the 
range.  It  can  be  fixed  within  narrower  limits  by 
inoculating  in  a  similar  manner  a  series  of  tubes  of 
bouillon  which  represent  smaller  variations  in  reaction 
than  those  previously  employed  (say,  i  instead  of  5) 
for  five  points  on  either  side  of  the  previously  observed 
optimum.  For  example,  the  optimum  reaction  ob- 
served in  the  set  of  experiments  to  determine  the 
range  was  + 10.  Now  plant  tubes  having  reactions 

of  +15,  +14,  +i3>  +I2>  +IJ>  +I0>  +9>  +8»  +7» 
+6,  +5,  and  observe  as  before. 

IV.  Resistance  to  Lethal  Agents. — 

(A)  Desiccation. — 

Apparatus  Required: 

Mueller's  desiccator.  This  consists  of  a  bell  glass  fitted  with  an 
exhaust  tube  and  stop-cock  (d),  which  can  be  secured  to  a  plate- 
glass  base  (c)  by  means  of  wax  or  grease.  It  contains  a  cylindrical 
vessel  of  porous  clay  (a)  into  the  top  of  which  pure  sulphuric  acid 
is  poured  whilst  the  material  to  be  dried  is  placed  within  its  walls 
on  a  glass  shelf  (6) .  The  air  is  exhausted  from  the  interior  and 
the  acid  rapidly  converts  the  clay  vessel  into  a  large  absorbing 
surface  (Fig.  157). 

Exhaust  pump. 

Pure  concentrated  sulphuric  acid. 

Sterile  cover-slips. 

Sterile  forceps. 

Culture  flask  containing  200  c.c.  nutrient  bouillon. 

Sterile  ventilated  Petri  dish.  This  is  prepared  by  bending  three 
short  pieces  of  aluminium  wire  into  V  shape  and  hanging  these  on 
the  edge  of  the  lower  dish  and  resting  the  lid  upon  them  (Fig.  1 58). 

METHOD. — 

1.  Prepare  a  sunace  cultivation  on  nutrient  agar  in 
a  culture  bottle  and  incubate  under  optimum  condi- 
tions for  forty-eight  hours. 

2 .  Examine  preparations  from  the  cultivation,  micro- 
scopically, to  determine  the  absence  of  spores. 

3.  Pipette  5  c.c.  sterile  normal  saline  solution  into 
the  flask  and  suspend  the  entire  growth  in  it. 


RESISTANCE    TO    LETHAL   AGENTS 


3°7 


4.  Spread   the   suspension   in   thin,    even   films   on 
sterile  cover-slips  and  deposit  inside  sterile  "plates" 
to  dry. 

5.  As  soon  as  dry,  transfer  the  cover-slip  films  to  the 
ventilated  Petri  dish  by  means  of  sterile  forceps. 


g« 


.•.;.-<.-si-.:;-.--:"mi~  £tf 


FIG.  157. — Mueller's  desiccator. 

6.  Place  the  Petri  dish  inside  the  Mueller's  desiccator; 
fill  the  upper  chamber  with  pure  sulphuric  acid,  cover 
with  the  bell  jar,  and  exhaust  the  air  from  its  interior. 


FIG.  158. — Petri  dish  for  drying  cultivations. 

Ten  minutes  later  connect  up  the  desiccator  to  a 
sulphuric  acid  wash-bottle  interposing  an  air  filter  so 
that  only  dry  sterile  air  enters. 


308  METHODS    OF   IDENTIFICATION    AND    STUDY 

7.  At  intervals  of  five  hours  open  the  apparatus, 
remove  one  of  the   cover-slip   films   from  the  Petri 
dish,  and  transfer  it  to  the  interior  of  a  culture  flask, 
with   every   precaution   against    contamination.     Re- 
seal  the  desiccator  and  again  exhaust,  and  subsequently 
admit  dry  sterile  air  as  before. 

8.  Incubate  the  culture  flask  under  optimum  condi- 
tions until  the  completion  of  seven  days,  if  necessary; 
and  determine  the  time  exposure  at  which  death  occurs. 

9.  Pour  plates  from  those  culture  flasks  which  grow, 
to  determine  the  absence  of  contamination. 

10.  Repeat  these  observations  at  hourly  intervals  for 
the   five  hours  preceding  and  succeeding  the  death 
time,  as  determined  in  the  first  set  of  experiments. 

(£)  Light.— 

(a)   Diffuse  Daylight: 

i.  Prepare  a  tube  cultivation  in  nutrient  bouillon, 
and  incubate  under  optimum  conditions,  for  forty-eight 
hours. 


FIG.  159.— Plate  with  star  for  testing  effect  of  light. 

2.  Pour  twenty  plate  cultivations,  ten  of  nutrient 
gelatine  and  ten  of  nutrient  agar,  each  containing  o.i 
c.c.  of  the  bouillon  culture. 

3.  Place  one  agar  plate  and  one  gelatine  plate  into 
the  hot  and  cold  incubators,  respectively,  as  controls. 

4.  Fasten  a  piece  of  black  paper,  cut  the  shape  of  a 
cross  or  star,  on  the  centre  of  the  cover  of  each  of  the 
remaining  plates  (Fig.  159). 


RESISTANCE    TO    LETHAL  AGENTS  309 

5.  Expose  these  plates  to  the  action  of  diffuse  day- 
light (not  direct  sunlight)  in  the  laboratory  for  one, 
two,  three,  four,  five,  six,  eight,  ten,  twelve  hours. 

6.  After  exposure  to  light,  incubate  under  optimum 
conditions. 

7.  Examine  the  plate  cultivations  after  twenty-four 
and  forty-eight  hours'  incubation,  and  compare  with 
the  two  controls.     Record  results.     If  growth  is  absent 
from  that  portion  of  the  plate  unprotected  by  the 
black  paper,  continue  the  incubation  and  daily  obser- 
vation until  the  end  of  seven  days. 

8.  Control  the  results. 

(b)  Direct  Sunlight : 

1.  Prepare    plate    cultivations    precisely   as   in   the 
former  experiments  and  place  the  two  controls  in  the 
incubators. 

2.  Arrange  the  remaining  plates  upon  a  platform  in 
the  direct  rays  of  the  sun. 

3.  On  the  top  of  each  plate  stand  a  small  glass  dish 
14  cm.  in  diameter  and  5  cm.  deep. 

4.  Fill  a  solution  of  potash  alum  (2  per  cent,  in  dis- 
tilled water)  into  each  dish  to  the  depth  of  2  cm.  to 
absorb  the  heat  of  the  sun's  rays  and  so  eliminate  possi- 
ble effects  of  temperature  on  the  cultivations. 

5.  After  exposures  for  periods  similar  to  those  em- 
ployed  in   the   preceding   experiment,    incubate   and 
complete  the  observation  as  above. 

(c)  Primary    Colours:     Each    colour — violet,    blue, 
green  and  red — must  be  tested  separately. 

1.  Prepare  plate  cultivations,   as    in    the    previous 
"light"  experiments,  and  incubate  controls. 

2.  Fasten  a  strip  of  black  paper,  3  cm.  wide,  across 
one  diameter  of  the  cover  of  each  plate. 

3.  Coat  the  remainder  of  the  surface  of  the  cover 
with  a  film  of  pure  photographic  collodion  which  con- 
tains 2  per  cent,  of  either  of  the  following  aniline  dyes, 
as  may  be  necessary. 


310  METHODS    OF   IDENTIFICATION   AND    STUDY 

Chrysoidin  (for  red) . 
Malachite  green  (for  green) . 
Eosin,  bluish  (for  blue) . 
Methyl  violet  (for  violet) . 

4.  Expose  the  plates,  thus  prepared,  to  bright  day- 
light (but  not  direct  sunlight)  for  varying  periods,  and 
complete  the  observations  as  in  the  preceding  experi- 
ments.    The  bactericidal  action  of  light  appears  to 
depend  upon  the  more  refrangible  rays  of  the  violet 
end  of  the  spectrum  and  is  noted  whether  the  red  yellow 
rays  are  transmitted  or  not. 

5.  Control  the  results. 

NOTE. — The  ultra-violet  rays  obtained  from  a  quartz  mercury 
vapour  lamp  destroy  bacterial  life  with  great  rapidity  under  labo- 
ratory conditions. 

(C)  Heat. — (Vide  Thermal  Death-point,  page  298.) 

(D)  Antiseptics    and   Disinfectants. — The  resistance 
exhibited  by  any  given  bacterium  toward  any  specified 
disinfectant  or  germicide  should  be  investigated  with 
reference  to  the  following  points: 

(A)  Inhibition  coefficient — i.   e.,   that  percentage  of 
the  disinfectant  present  in  the  nutrient  medium  which 
is  sufficient  to  prevent  the  growth  and  multiplication 
of  the  bacterium. 

(B)  Inferior  lethal  coefficient — i.  e.,  the  time  expo- 
sure necessary  to  kill  vegetative  forms  of  the  bacterium 
suspended  in  water  at  20°  to  25°  C.,  in  which  the  disin- 
fectant is  present  in  medium  concentration   (concen- 
tration insufficient  to  cause  plasmolysis) .     And  if  the 
bacterium  is  one  which  forms  spores, 

(C)  Superior  lethal  coefficient — i.  e.,  the  time  expo- 
sure necessary  to  kill  the  spores  of  the  bacterium  under 
conditions  similar  to  those  obtaining  in  B. 

The  example  here  detailed  only  specifically  refers 
to  certain  of  the  disinfectants : 

viz:-  Bichloride  of  mercury; 


INHIBITION    COEFFICIENT  311 

Formaldehyde ; 

Carbolic  acid; 

investigated  with  regard  to  B.  anthracis,  but  the  tech- 
nique is  practically  similar  for  all  other  chemical 
disinfectants. 

Inhibition  Coefficient. — 

Apparatus  Required: 

Case  of  sterile  pipettes,  10  c.c.  (in  tenths). 

Case  of  sterile  pipettes,  i  c.c.  (in  tenths). 

Sterile  tubes  or  capsules  for  dilutions. 

Tubes  of  nutrient  bouillon  each  containing  a  measured  10  c.c. 
of  medium. 

Twenty-four-hour-old  agar  culture  of  a  recently  isolated  B. 
anthracis 

Germicides: 

1.  Five  per  cent,  aqueous  solution  of  carbolic  acid. 

2.  One  per  cent,  aqueous  solution  of  perchloride  of  mercury. 

3.  One- tenth  per  cent,  aqueous  solution  of  formaldehyde. 

METHOD. — 

1.  Number  six  bouillon  tubes  consecutively  i  to  6. 
Inoculate  each  from  the  stock  cultivation  of  B.  anthra- 
cis and  at  once  add  varying  quantities1  of  the  carbolic 
acid  solution,  viz. : 

To  tube  i  add  2  .  o  c.c.  ( =  i :  100) 
To  tube  2  add  i . o  c.c.  ( =  i :  200) 
To  tube  3  add  o. 6  c.c.  ( =  i  1300) 
To  tube  4  add  o .  5  c.c.  ( =  i :  400) 
To  tube  5  add  0.4  c.c.  ( =  i :  500) 
To  tube  6  add  o .  2  c.c.  ( =  i :  i  ,000) 

2.  Prepare    a    similar    series    of    tube    cultivations 
numbered   consecutively    7    to    12    and   add    varying 
quantities  of  the  mercuric  perchloride  solution,  viz. : 

To  tube  7  add  o.i  (=1:1,000) 
To  tube  8  add  0.05  (=1:2,000) 
To  tube  9  add  0.03  (  =  1:3,000) 
To  tube  10  add  0.025  (=i-'4,00°) 
To  tube  1 1  add  o .  02  ( =  i :  5,000) 
To  tube  12  add  o.o i  (=1:10,000) 

1  The  quantities  here  given  are  not  absolutely  correct.  If  exactitude  is 
essential  the  student  must  calculate  the  amount  required  by  the  aid  of  the 
Percentage  Formula,  Appendix,  page  496. 


312  METHODS    OF   IDENTIFICATION   AND    STUDY 

3.  Prepare  a  similar  series  of  tube  cultivations 
numbered  consecutively  13  to  18  and  add  varying 
quantities  of  the  formaldehyde  solution,  viz. : 


To  tube  No.  13  add  i  .o  c.c.  (  = 
To  tube  No.  14  add  0.4  c.c.  (  = 
To  tube  No.  15  add  0.2  c.c.  (  = 
To  tube  No.  16  add  o.  i  c.c.  (  = 
To  tube  No.  17  add  0.075  c-c-  (  = 
To  tube  No.  18  add  0.05  c.c.  (  = 


1,000) 

2,5°°) 

5,ooo) 

10,000) 

15,000) 

20,000) 


4.  Incubate  all  three  sets  of  cultivations  under  opti- 
mum conditions  as  to  temperature  and  atmosphere. 

5.  Examine  each  of  the  culture  tubes  from  day  to 
day,  until  the  completion  of  seven  days,   and  note 
those  tubes,  if  any,  in  which  growth  takes  place. 

6.  From  such  tubes  as  show  growth  prepare  sub- 
cultivations  upon  suitable  media,  and  ascertain  that 
the   organism   causing   the   growth   is   the   one   orig- 
inally employed  in  the  test   and   not   an   accidental 
contamination. 

Inferior  Lethal  Coefficient. — 

Apparatus  Required: 

Highly  concentrated  solutions  of  the  disinfectants. 

Sterile  test-tubes  in  which  to  make  dilutions  from  the  concen- 
trated solutions  of  the  disinfectants. 

Hanging-drop  slides. 

Cover-slips. 

Erlenmeyer  flask  containing  100  c.c.  sterile  distilled  water. 

Case  of  sterile  pipettes,  10  c.c.  (in  tenths  of  a  cubic  centimetre). 

Case  of  sterile  pipettes,  i  c.c.  (in  tenths  of  a  cubic  centimetre). 

METHOD. — 

1.  Prepare    a    surface    cultivation    of    the    "test" 
organism  B.  anthracis  upon  nutrient  agar  in  a  culture 
bottle   and   incubate  under  optimum   conditions   for 
twenty-four  hours ;  then  examine  the  cultivation  micro- 
scopically to  determine  the  absence  of  spores. 

2.  Prepare  solutions  of  different  percentages  of  each 
disinfectant. 

3.  Make  a  series  of  hanging-drop  preparations  from 


SUPERIOR    LETHAL    COEFFICIENT  313 

the  agar  culture,  using  a  loopful  of  disinfectant  solu- 
tion of  the  different  percentages  to  prepare  the  emulsion 
on  each  cover-slip. 

4.  Examine  microscopically  and  note  the  strongest 
solution  which  does  not  cause  plasmolysis  and  the 
weakest  solution  which  does  plasmolyse  the  organism. 

5.  Make  control  preparations  of  these  two  solutions 
and  determine  the  percentage  to  be  tested. 

6.  Pipette  10  c.c.  sterile  water  into  the  culture  bottle 
and  suspend  the  entire  surface  growth  in  it. 

7.  Transfer  the  suspension  to  the  Erlenmeyer  flask 
and  mix  it  with  the  90  c.c.  of  sterile  water  remaining 
in  the  flask. 

8.  Pipette  10  c.c.  of  the  diluted  suspension  into  each 
of  ten  sterile  test-tubes. 

9.  Label  one  of  the  tubes  "Control"  and  place  it 
in  the  incubator  at  18°  C. 

10.  Add  to  each  of  the  remaining  tubes  a  sufficient 
quantity J  of  a  concentrated  solution  of  the  disinfectant 
to  produce  the  percentage  previously  determined  upon 
(vide  step  5). 

11.  Incubate  the  tubes  at  18°  C.  to  20°  C. 

12.  At   hourly  intervals  remove  the   control  tube 
and  one  of  the  tubes  with  added  disinfectant  from  the 
incubator. 

13.  Make  a  subcultivation  from  both  the  control  and 
the  test  suspension,  upon  the  surface  of  nutrient  agar; 
incubate  under  optimum  conditions. 

14.  Observe  these  culture  tubes  from  day  to  day 
until  the  completion  of  seven  days,   and  determine 
the  shortest  exposure  necessary  to  cause  the  death  of 
vegetative  forms. 

Superior  Lethal  Coefficient.— 

i.  Prepare  surface  cultivations  of  the  "test"  organ- 
isms upon  nutrient  agar  in  a  culture  bottle,  and  incu- 

1  See  Percentage  Formula,  Appendix,  page  496. 


314  METHODS    OF   IDENTIFICATION   AND    STUDY 

bate  under  optimum  conditions,  for  three  days,   for 
the  formation  of  their  spores. 

2.  Transfer  the  emulsion  to  a  sterile  test-tube  and 
heat  in  the  differential  steriliser  for  ten  minutes  at 
80°  C.  to  destroy  all  vegetative  forms. 

3 .  Employing  that  percentage  solution  of  the  disinfec- 
tant determined  in  the  previous  experiment,  and  com- 
plete the  investigations  as  detailed  therein,  steps  7  to 
14,  increasing  the  interval  between  planting  the  sub- 
cultivations  to  two,  three,  or  five  hours  if  considered 
advisable. 

NOTE. — Where  it  is  necessary  to  leave  the  organisms  in  contact 
with  a  strong  solution  of  the  disinfectant  for  lengthy  periods, 
some  means  must  be  adopted  to  remove  every  trace  of  the  disin- 
fectant from  the  bacteria  before  transferring  them  to  fresh  culture 
media;  otherwise,  although  not  actually  killed,  the  presence  of  the 
disinfectant  may  prevent  their  development,  and  so  give  rise  to 
an  erroneous  conclusion.  Consequently  it  is  essential  in  all 
germicidal  experiments  to  determine  first  of  all  the  inhibition 
coefficient  of  the  germicide  employed.  Under  the  circumstances 
referred  to  above  it  is  usually  sufficient  to  prepare  the  subcultures 
in  such  a  volume  of  fluid  nutrient  medium  as  would  suffice  to 
reduce  the  concentration  of  the  germicide  to  about  one  hun- 
dredth of  the  inhibition  percentage,  assuming  that  the  entire 
bulk  of  inoculum  was  made  up  of  that  strength  of  germicide 
employed  in  the  test.  In  some  cases  it  is  a  simple  matter  to  neu- 
tralise the  germicide  and  render  it  inert  by  washing  the  organisms 
in  some  non -germicidal  solution  (such  for  example  as  ammonium 
sulphide  when  using  mercurial  salts  as  the  germicide).  When, 
however,  it  is  desired  to  remove  the  last  traces  of  germicide  proceed 
as  follows: 

1.  Transfer  the  suspension  of  bacteria  to  sterile  centrifugal 
tubes;  add  the  required  amount  of  disinfectant,  and  allow  it  to 
remain  in  contact  with  the  bacteria  for  the  necessary  period. 

2.  Centrifugalise thoroughly,  pipette  off  the  supernatant  fluid; 
fill  the  tube  with  sterile  water  and  distribute  the  deposit  evenly 
throughout  the  fluid. 

3.  Centrifugalise  again,  pipette  off  the  supernatant  fluid;  fill 
the  tube  with  sterile  water;  distribute  the  deposit  evenly  through- 
out the  fluid,  and  transfer  the  suspension  to  a  litre  flask. 

4.  Make  up  to  a  litre  by  the  addition  of  sterile  water;  filter  the 
suspension  through  a  sterile  porcelain  candle. 

5.  Emulsify  the  bacterial  residue  with  5  c.c.  sterile  bouillon. 

6.  Prepare  the  necessary  subcultivations  from  this  emulsion. 


PATHOGENETIC    PROPERTIES  315 

PATHOGENESIS. 

Living  Bacteria. — 

(a)  Psychrophilic  Bacteria :  When  the  organism  will 
only  grow  at  or  below  18°  to  20°  C., 

1.  Prepare  cultivations  in  nutrient  broth  and  in- 
cubate under  optimum  conditions. 

2.  After  seven  days'  incubation  inject  that  amount 
of  the   culture  corresponding  to    i    per  cent,   of  the 
body-weight  of  a  healthy  frog,  into  the  reptile's  dorsal 
lymph  sac.. 

3.  Observe  until  death  takes  place,  or,  in  the  event 
of  a  negative  result,  until  the  completion  of  twenty- 
eight  days  (vide  Chapter  XVIII). 

4.  If,  and  when,  death  occurs,  make  a  careful  post- 
mortem examination  (vide  Chapter  XIX) . 

(b)  Mesophilic  Bacteria:  When  the  organism  grows 
at  35°  to  37°  C, 

1.  Prepare  cultivations  in  nutrient  broth  and  incu- 
bate under  optimum  conditions  for  forty-eight  hours. 

2.  Select  two  white  mice,  as  nearly  as  possible  of 
the  same  age,  size,  and  weight. 

3.  Inoculate    the    first    mouse,    subcutaneously    at 
the  root  of  the  tail,  with  an  amount  of  cultivation 
equivalent  to  i  per  cent,  of  its  body- weight. 

4.  Inoculate    the    second    mouse    intraperitoneally 
with  a  similar  dose. 

5.  Observe  carefully  until  death  occurs,  or  until  the 
lapse  of  twenty-eight  days. 

6.  If  the  inoculated  animals  succumb,  make  com- 
plete post-mortem  examination. 

If  death  follows  shortly  after  the  injection  of  cul- 
tivations of  bacteria,  the  inoculation  experiments 
should  be  repeated  two  or  three  times.  Then,  if  the 
organism  under  observation  invariably  exhibits  patho- 
genic effects,  steps  should  be  taken  to  ascertain,  if 
possible,  the  minimal  lethal  dose  (vide  infra)  of  the 
growth  upon  solid  media  for  the  frog  or  white  mouse 


316  METHODS    OF   IDENTIFICATION  AND    STUDY 

respectively.  Other  experimental  animals — e.  g.,  the 
white  rat,  guinea-pig,  and  rabbit — should  next  be 
tested  in  a  similar  manner. 

7.  If  the  inoculated  mice  are  unaffected,  test  the 
action  of  the  organism  in  question  upon  white  rats, 
guinea-pigs,  rabbits,  etc. 

Minimal  Lethal  Dose  (m.  I.  d.);  If  the  purpose  of  the 
inoculation  is  to  determine  the  minimal  lethal  dose,  a 
slightly  different  procedure  must  be  followed.  For  this 
and  other  exact  experiments  a  special  platinum  loop  is 
manufactured,  some  2.5  mm.  by  0.75  mm.,  with  parallel 
sides,  and  calibrated  by  careful  weighing,  to  determine 
approximately  the  amount  of  moist  bacterial  growth 
the  loop  will  hold  when  filled. 

1.  The   cultivation  must   be   prepared   on   a   solid 
medium  of  the  optimum  reaction,  incubated  at  the 
optimum  temperature,  and  injected  at  the  period  of 
greatest  activity  and  vigour,  of  the  particular  organism 
it  is  desired  to  test. 

2.  Arrange  four  sterile  capsules  in  a  row  and  label 
them  I,  II,  III,  and  IV.     Into  the  first  deliver  10  c.c. 
sterile  bouillon  by  means  of  a  sterile  graduated  pipette ; 
and  into  each  of  the  remaining  three,  9.9  c.c. 

3.  Remove  one  loopful  of  the  bacterial  growth  from 
the  surface  of  the  medium  in  the  culture  tube,  observ- 
ing the  usual  precautions  against  contamination,  and 
emulsify  it  evenly  with  the  bouillon  in  the  first  capsule. 
Each  cubic  centimetre  of  the  emulsion  will  now  con- 
tain   one-tenth   of   the   organisms    contained   in    the 
original  loopful  (written  shortly  o.i  loop). 

4.  Remove  o.i  c.c.  of  the  emulsion  in  the  first  cap- 
sule  by  means   of   a   sterile   graduated   pipette   and 
transfer  it  to  the  second  capsule  and  mix  thoroughly. 
Drop  the  infected  pipette  into  a  jar  of  lysol  solution. 
This  makes  up  the  bulk  of  the  fluid  in  the  second  cap- 
sule to  10  c.c.,  and  therefore  every  cubic  centimetre 
of  bouillon  in  capsule  II  contains  o.ooi  loop. 


MINIMAL    LETHAL   DOSE  317 

5.  Similarly,  o.i  c.c.  of  the  mixture  is  transferred 
from  capsule  II  to  capsule  III  (i  c.c.  of  bouillon  in 
capsule  III  contains  o.ooooi  loop),  and  then  from 
capsule  III  to  capsule  IV  (i  c.c.  of  bouillon  in  capsule 
IV  contains  o.ooooooi  loop). 

The  dilutions  thus  prepared  may  be  summarised  in 
a  table; 

Capsule      I=i  loopful+io  c.c.  water  .*.  i  c.c.  =  o.i  loop. 

Capsule    II  =  o.i  c.c.  capsule  I     +9.9  c.c.  water  .'.  i  c.c.  =  o.ooi  loop. 

Capsule  III  =  o.i  c.c.  capsule  II  +9.9  c.c.  water  .'.  i  c.c.  =  0.00001  loop. 

Capsule  IV  =  o.i  c.c.  capsule  III  +  9.9  c.c.  water  .'.  i  c.c.  =  0.0000001  loop. 


6.  With  sterile  graduated  pipettes  remove  the  neces- 
sary quantity  of  bouillon  corresponding  to  the  various 
divisors  of  ten  of  the  loop  from  the  respective  capsules, 
and  transfer  each  "dose"  to  a  separate  sterile  capsule 
and  label;  and  to  such  doses  as  are  small  in  bulk,  add 
the  necessary  quantity  of  sterile  bouillon  to  make  up 
to  i  c.c. 

7.  Multiples  of  the  loop  are  prepared  by  emulsifying 
i,  2,  5,  or  10  loops  each  with  i  c.c.  sterile  bouillon  in 
separate  sterile  capsules. 

8.  Inoculate  a  series  of  animals  with  these  measured 
doses,  filling  the  syringe  first  from  that  capsule  con- 
taining the  smallest  dose,  then  from  the  capsule  con- 
taining the  next  smallest,  and  so  on.     If  care  is  taken, 
it  will  not  be  found  necessary  to  sterilise  the  syringe 
during  the  series  of  inoculations. 

9.  Plant  tubes  of  gelatine  or  agar,  liquefied  by  heat, 
from  each  of  the  higher  dilutions,  say  from  o.ooooooi 
loop  to  o.o  i  loop;  pour  plates  and  incubate.     When 
growth  is  visible  enumerate  the  number  of  organisms 
present  in  each,  average  up  and  calculate  the  number 
of  bacteria  present  in  one  loopful  of  the  inoculum. 

10.  The  smallest  dose  which  causes  the  infection  and 
death  of  the  inoculated  animal  is  noted  as  the  minimal 
lethal  dose. 


318  METHODS    OF   IDENTIFICATION  AND    STUDY 

Toxins. — 

Prepare  flask  cultivations  of  the  organism  under 
observation  in  glucose  formate  broth,  and  incubate  for 
fourteen  days  under  optimum  conditions. 

(a)   Intracellular  or  Insoluble  Toxins : 

1.  Heat  the  fluid  culture  in  a  water-bath  at  60°  C. 
for  thirty  minutes.     (The  resulting  sterile,  turbid  fluid 
is  often  spoken  of  as  "killed"  culture,) 

2.  Inoculate  a  tube  of  sterile  bouillon  with  a  similar 
quantity,    and    incubate  under  optimum   conditions. 
This  " control"  then  serves  to  demonstrate  the  freedom 
of  the  toxin  from  living  bacteria. 


FIG.  160. — Apparatus  arrange  for  toxin  filtration. 

3.  Inject  intraveneously  that  amount  of  the  culti- 
vation corresponding  to  i  per  cent,  of  the  body- weight 
of  the  selected  animal,  usually  one  of  the  small  rodents. 

4.  Observe  during  life  or  until  the  completion  of 
twenty-eight  days,  and  in  the  event  of  death  occurring 
during  that  period,  make  a  complete  post-mortem  ex- 
amination. 

5.  Repeat  the  experiment  at  least  once.     In  the 
event  of  a  positive  result  estimate  the  minimal  lethal 
dose  of   "killed"  culture  for  each  of  the  species  of 
animals  experimented  upon. 

(b)  Extracellular  or  Soluble  Toxins : 


MINIMAL    LETHAL    DOSE  319 

1.  Filter  the  cultivation  through  a  porcelain  filter 
candle  (Berkfeld)  into  a  sterile  filter  flask,  arranging 
the  apparatus  as  in  the  accompanying  figure  (Fig.  160). 

2.  Inoculate   mice,    rats,    guinea-pigs,    and   rabbits 
subcutaneously   with   that   quantity   of   toxin   corre- 
sponding to  i  per  cent,  of  the  body- weight  of  each 
respectively,  and  observe,  if  necessary,  until  the  com- 
pletion of  one  month. 

3.  Inoculate  a  " control"  tube  of  bouillon  with  a 
similar  quantity  and  incubate,  to  determine  the  freedom 
of  the  filtered  toxin  from  living  bacteria. 

4.  In  the  event  of  a  fatal  termination  make  com- 
plete and  careful  post-mortem  examinations. 

5.  Repeat  the  experiments  and,  if  the  results  are 
positive,  ascertain  the  minimal  lethal  dose  of  toxin 
for  each  of  the  susceptible  animals. 

The  estimation  of  the  m.  I.  d.  of  a  toxin  is  carried 
out  on  lines  similar  to  those  laid  down  for  living  bac- 
teria (vide  page  316)  merely  substituting  i  c.c.  of  toxin 
as  the  unit  in  place  of  the  unit  "loopful"  of  living 
culture. 

It  frequently  happens,  during  the  course  of  casual  in- 
vestigations that  a  bouillon-tube  culture  is  available 
for  a  toxin  test  whilst  a  flask  cultivation  is  not.  In 
such  cases,  Martin's  small  filter  candle  and  tube  (Fig. 
161)  specially  designed  for  the  filtration  of  small  quan- 
tities of  fluid,  is  invaluable.  This  consists  of  a  narrow 
flilter  flask  just  large  enough  to  accommodate  an  ordi- 
nary 18X2  cm.  test-tube.  The  mouth  of  the  tubular 
Chamberland  candle  15X1.5  cm.  is  closed  by  a  perfor- 
ated rubber  cork  into  which  fits  the  end  of  the  stem 
of  a  thistle  headed  funnel,  whilst  immediately  below 
the  butt  of  the  funnel  is  situated  a  rubber  cork  to  close 
the  mouth  of  the  filter  flask.  When  the  apparatus  is 
fixed  in  position  and  connected  to  an  exhaust  pump, 
the  cultivation  is  poured  into  the  head  of  the  funnel 
and  owing  to  the  relatively  large  filtering  surface  the 


320 


METHODS    OF   IDENTIFICATION   AND    STUDY 


germ  free  filtrate  is  rapidly  drawn  through  into  the 
test-tube  receiver. 

Raising  the  Virulence   of   an   Organism. — If   it   is 

desired  to  raise  or  "exalt"  the  virulence  of  a  feebly 
pathogenic  organism,  special  methods  of  inoculation 
are  necessary,  carefully  adjusted  to  the  exigencies  of 
each   individual   case.     Among  the 
most  important  are  the  following : 

1 .  Passage  of  Virus. — The  inocula- 
tion of  pure  cultivations  of  the  organ- 
ism into  highly  susceptible  animals, 
and  passing  it  as  rapidly  as  possible 
from  animal  to  animal,  always  select- 
ing that   method    of   inoculation — 
e.  g.,  intraperitoneal — which  places 
the  organism  under  the  most  favor- 
able conditions  for  its  growth  and 
multiplication. 

2 .  Virus  Plus  Virulent  Organisms. 
— The  inoculation  of  pure  cultiva- 
tions of  the  organism  together  with 
pure  cultivations  of  some  other  mi- 
crobe which  in  itself  is  sufficiently 
virulent  to  ensure  the  death  of  the 
experimental  animal,  either  into  the 
same  situation  or  into  some  other 

for  small  quantities  part  of  the  body.  By  this  associa- 
of  fluid.  \  ... 

tion   the  organism  of  low  virulence 

will  frequently  acquire  a  higher  degree  of  virulence, 
which  may  be  still  further  raised  by  means  of  "pas- 
sages" (vide  supra). 

3.  Virus  Plus  Toxins. — The  inoculation  of  pure 
cultivations  of  the  organism  into  some  selected  situa- 
tion, together  with  the  subcutaneous,  intraperitoneal, 
or  intravenous  injection  of  a  toxin — e.  g.,  one  of  those 
elaborated  by  the  proteus  group — either  simultane- 
ously with,  before,  or  immediately  after,  the  injection 


ATTENUATING    THE    VIRULENCE    OF   AN    ORGANISM   321 

of  the  feeble  virus.  By  this  means  the  natural  resist- 
ance of  the  animal  is  lowered,  and  the  organism  inocu- 
lated is  enabled  to  multiply  and  produce  its  pathogenic 
effect,  its  virulence  being  subsequently  exalted  by 
means  of  "passages." 

Attenuating  the  Virulence  of  an  Organism. — Attenu- 
ating or  lowering  the  virulence  of  a  pathogenic  microbe 
is  usually  attained  with  much  less  difficulty  than  the 
exaltation  of  its  virulence,  and  is  generally  effected 
by  varying  the  environment  of  the  cultivations,  as 
for  example: 

1.  Cultivating  in  such  media  as  are  unsuitable  by 
reason  of  their  (a)  composition  or  (b)  reaction. 

2 .  Cultivating  in  suitable  media,  but  at  an  unsuitable 
temperature. 

3 .  Cultivating  in  suitable  media,  but  in  an  unsuitable 
atmosphere. 

4.  Cultivation  in  suitable  media,  but  under  unfavor- 
able conditions  as  to  light,  motion,  etc. 

Attenuation  of  the  virus  can  also  be  secured  by 

5.  Passage  through  naturally  resistant  animals. 

6.  Exposure  to  desiccation. 

7.  Exposure  to  gaseous  disinfectants. 

8.  By  a  combination  of  two  or  more  of  the  above 
methods. 

IMMUNISATION. 

The  further  study  of  the  pathogenetic  powers  of  any 
particular  bacterium  involves  the  active  immunisation 
of  one  or  more  previously  normal  animals.  This  end  may 
be  attained  by  various  means ;  but  it  must  be  remem- 
bered that  immunisation  is  not  carried  out  by  any  hard 
and  fast  rule  or  by  one  method  alone,  but  usually  by  a 
combination  of  methods  adapted  to  the  exigencies  of 
each  particular  case.  The  ordinary  methods  include : 

A.  Active  Immunisation. 

I.   By  inoculation  with  dead  bacteria  (i.  e., 


322  METHODS    OF   IDENTIFICATION  AND    STUDY 

bacteria  killed   by  heat;  the  action  of 
ultra-violet  rays,  of  chemical  germicides, 
or  by  autolysis). 
II.  By  the  inoculation  of  attenuated  strains 

of  bacteria'. 

III.  By  the  inoculation  of  living  virulent 
bacteria  (exalted  in  virulence  if  neces- 
sary). 

B.  Combined  Active  and  Passive  Immunisation : 
IV.  By   the   inoculation  of    toxin-antitoxin 
mixtures. 

ACTIVE  IMMUNISATION. 

The  immunisation  of  the  rabbit  against  the  Diplococ- 
cus  pneumoniae  may  be  instanced  as  an  example  of  the 
general  methods  of  immunisation  of  laboratory  animals. 

1.  Take  a  full  grown  rabbit  weighing  not  less  than 
1 200  to  1500  grammes  (large  rabbits  of  2000  grammes 
and  over  are  the  most  suitable  for  immunising  experi- 
ments).    Observe  weight  and  temperature   carefully 
during  the  few  days  occupied  in  the  following  steps. 

2.  Inoculate   a  small  rabbit  intraperitoneally  with 
one  or  two  loopfuls  of  a  twenty-four-hour-old  blood 
agar  cultivation  of  a  virulent  strain  of  Diplococcus 
pneumoniae. 

Death  should  follow  within  twenty-four  hours,  and  in 
any  case  will  not  be  delayed  beyond  forty-eight  hours. 

3.  Under  aseptic  precautions,  at  the  post-mortem, 
transfer  a  loopful  of  heart  blood  to  an  Erlenmeyer  flask 
containing  50  c.c.  sterile  nutrient  broth.     Incubate  at 
37°  C.  for  twenty-four  hours. 

4.  Prepare  also  several  blood  agar  cultures  from  the 
heart  blood  of  the  rabbit,  label  them  all  O.C.  (original 
culture) .     After  twenty-four  hours  incubation  at  3  7°  C. 
place  an  india-rubber  cap  over  the  plugged  mouth  of 
the  tube  of  all  but  one  of  these  cultures  and  paint  the 
cap  with  Canada  balsam  or  shellac  varnish,  dry,  and 
replace  in  the  hot  incubator. 


ACTIVE    IMMUNISATION  323 

This  will  prevent  evaporation,  and  cultures  thus 
sealed  will  remain  unaltered  in  virulence  for  a  consider- 
able time. 

5.  Make  a  fresh  subcultivation  on  blood  agar  from 
the  uncapped  O.  C.  cultivation  and  after  twenty-four 
hours   incubation  at   37°  C.   determine   the   minimal 
lethal  dose  of  this  strain  upon  a  series  of  mice  (see 
page  3 1 6). 

6.  Suspend   the    flask  containing  the  twenty-four- 
hour-old  broth  culture  (step  3)  in  the  water-bath  at 
60°  C.  for  one  hour.     Cool  the  flask  rapidly  under  a 
stream  of  cold  water. 

7.  Determine  the  sterility  of  this  (?)  killed  cultiva- 
tion by  transferring  one  cubic  centimetre  to  each  of 
several  tubes  of  nutrient  broth,  and  incubate  at  37°  C. 
for    twenty-four    hours.     If    growth    of    Diplococcus 
pneumonise  occurs,  again  heat  culture  in  water- bath 
at  60°  C.  for  one  hour  and  again  test  for  sterility. 

8.  Inject  the  selected  rabbit  intravenously  (see  page 
363)  with  2  c.c.  of  the  killed  cultivation,  and  inject  a 
further  10  c.c.  into  the  peritoneal  cavity. 

During  the  next  few  days  the  animal  will  lose  some 
weight  and  perhaps  show  a  certain  amount  of  pyrexia. 

9.  When  the  temperature  and  weight  have  again 
returned  to  normal — generally  about  seven  days  after 
the  inoculation — again  inject  killed  cultivation,   this 
time  giving  a  dose  of  5  c.c.  intravenously  and  20  c.c. 
intraperitoneally.     A  temperature  and  weight  reaction 
similar  to,   but  less  marked  than  that  following  the 
first  injection  will  probably  be  observed,    but  after 
about  a  week's  interval  the  animal  will  be  ready  for  the 
next  injection. 

10.  When  ready  to  give  the  third  injection  prepare 
a  fresh  blood  agar  subculture  from  another  O.  C.  tube 
and  after  twenty-four    hours    incubation    prepare    a 
minimal  lethal  dose  (as  determined  in  5)  and  inject-  it 
subcutaneously  into  the  rabbit's  abdominal  wall. 


324  METHODS    OF   IDENTIFICATION   AND    STUDY 

A  slight  local  reaction  will  probably  be  observed  as 
well  as  the  weight  and  temperature  reactions. 

1 1.  A  week  to  ten  days  later  inject  a  similar  minimal 
lethal  dose  into  the  peritoneal  cavity. 

12.  Observe   the   weight   and   temperature    of   the 
rabbit   very    carefully,    and   regulating   the   dates   of 
inoculation  by  the  animal's  general  condition,  continue 
to    inject    living    cultivations    of    the    pneumococcus 
into  the  peritoneal  cavity,   gradually  increasing  the 
dose  by  multiples  of  ten. 

13.  At  intervals  of  two  months  samples  of  blood  may 
be  collected  from  the  posterior  auricular  vein  and  the 
serum  tested  for  specific  anti-bodies. 

14.  Under  favourable  conditions  it  will  be  found 
after  some  six  months  steady  work  that  the  rabbit 
may  be  injected  intraperitoneally  with  an  entire  blood 
agar  cultivation  without  any  ill  effects  being  apparent ; 
and  this  characteristic — resistance  to  the  lethal  effects 
of  large  doses  of  the  virus — is  the  sole  criterion  of 
immunity.     Further,  the  serum  separated  from  blood 
withdrawn  from  the  animal  about  a  week  after  an 
injection,  if  used  in  doses  of  .01  c.c.,  will  protect  a 
mouse  against  the  lethal  effects  of  at  least  ten  minimal 
lethal  doses  of  living  pneumococci. 

In  the  foregoing  illustration  it  has  been  assumed  that 
complete  acquired  active  immunity  has  been  conferred 
upon  the  experimental  rabbit  in  consequence  of  the  for- 
mation of  antibody,  specific  to  the  diplococcus  pneu- 
moniac,  sufficient  in  amount  to  ensure  the  destruction 
of  enormous  doses  of  the  living  cocci — the  antigen  (that 
is  the  substance  injected  in  response  to  which  antibody 
has  been  elaborated)  in  this  particular  case  being  the 
bacterial  protoplasm  of  the  pneumococcus  with  its 
endo- toxins. 

But  provided  death  does  not  immediately  follow  the 
injection  of  the  antigen,  specific  antibody  is  always 
formed  in  greater  or  lesser  amount;  and  in  experi- 


ACTIVE  IMMUNISATION  325 

mental  work  a  sufficient  amount  of  any  required  anti- 
body can  often  be  obtained  without  carrying  the  process 
of  immunisation  to  its  logical  termination. 

For  instance,  if  the  immunisation  of  a  rabbit  toward 
Bacillus  typhosus  is  commenced  on  the  lines  already 
set  out  it  will  often  be  found,  after  a  few  injections  of 
"killed"  cultivation  that  the  blood  serum  of  the  animal 
(even  when  diluted  with  several  hundred  times  its 
volume  of  normal  saline)  contains  specific  agglutinin 
for  B.  typhosus — and  if  the  sole  object  of  the  experi- 
ment has  been  the  preparation  of  agglutinin  the  inocu- 
lations may  well  be  stopped  at  this  point,  although  the 
animal  is  not  yet  immune  in  the  strict  meaning  of  the 
word. 

Again,  antibodies  may  be  formed  in  response  to 
antigens  other  than  infective  particles — thus  the  injec- 
tion into  suitable  animals  of  foreign  proteins  such  as 
egg  albumin,  heterologous  blood  sera  or  red  blood  discs 
from  a  different  species  of  animal,  will  result  in  the 
formation  of  specific  antibodies  possessing  definite 
affinities  for  their  respective  antigens. 

The  most  important  antibody  of  this  latter  type  is 
Haemolysin,  a  substance  that  makes  its  appearance  in 
the  blood  serum  of  an  animal  previously  injected  with 
washed  blood  cells  from  an  animal  of  a  different  species. 
The  serum  from  such  an  animal  possesses  the  power  of 
disintegrating  red  blood  discs  of  the  variety  employed 
as  antigen  and  causing  the  discharge  of  their  contained 
haemoglobin,  and  is  specific  in  its  action  to  the  extent 
of  failing  to  exert  any  injurious  effect  upon  the  red 
blood  cells  of  any  other  species  of  animal. 

The  action  of  this  serum  is  due  to  the  presence  of 
two  distinct  bodies,  complement  and  haemolysin. 

Complement  (or  alexine)  is  a  thermo-labile  readily 
oxidised  body  present  in  variable  but  unalterable 
amount  in  the  normal  serum  of  every  animal.  It  is 
a  substance  which  exerts  a  lytic  effect  upon  all  foreign 


326  METHODS    OF   IDENTIFICATION  AND    STUDY 

matter  introduced  into  the  blood  or  tissues;  but  by 
itself  is  a  comparatively  inert  body,  and  is  only  capable 
of  exerting  its  maximum  lytic  effect  in  the  presence  of 
and  in  combination  with  a  specific  antibody,  or  immune 
body. 

Complement  is  obtained  (unmixed  with  antibody)  by 
collecting  fresh  blood  serum  from  any  healthy  normal 
(that  is  uninoculated)  animal.  Guinea-pigs'  serum  is 
that  most  frequently  employed  for  experimental  work. 

Hcemolysin  (immune  body,  copula,  sensitising  body, 
amboceptor)  is  a  thermostable  antibody  formed  in 
response  to  the  injection  of  red  cells  which  although 
in  itself  inert  is  capable  of  linking  up  complement 
present  in  the  normal  serum  to  the  red  cells  of  the 
variety  used  as  antigen — a  combination  resulting  in 
haemolysis. 

Haemolysin  is  obtained  by  collecting  fresh  blood  serum 
from  a  suitably  inoculated  animal  and  exposing  it  to  a 
temperature  of  56°  C.  (to  destroy  the  thermo-labile 
complement)  for  15  to  30  minutes  before  use.  It  is 
then  referred  to  as  inactivated,  and  is  reactivated  by 
the  addition  of  fresh  normal  serum — that  is  serum 
containing  complement. 

Haemolysin  is  of  importance  academically  owing  to 
the  fact  that  many  of  the  problems  of  immunity  have 
been  elucidated  by  its  aid;  but  its  present  practical 
importance  lies  in  the  application  of  the  hcemolytic 
system  (that  is  haemolysin,  corresponding  erythrocyte 
solution  and  complement)  to  certain  laboratory 
methods  having  for  their  object  either  the  identifi- 
cation of  the  infective  entity  or  the  diagnosis  of  the 
existence  of  infection. 

For  use  in  these  laboratory  methods  of  diagnosis  it  is 
most  convenient  to  prepare  haemolytic  serum  specific  for 
human  blood — whether  the  laboratory  is  isolated  or 
attached  to  a  large  hospital.  Ox  blood,  sheep  blood  or 
goat  blood  if  readily  obtainable,  may  however  be  used 


H^EMOLYTIC    SERUM  327 

instead,  and  although  the  following  method  is  directed 
to  the  preparation  of  human  haemolysin  the  same 
procedure  serves  in  all  cases. 

THE  PREPARATION  OF  H^EMOLYTIC  SERUM. 

Apparatus  Required: 

Small  centrifuge,  preferably  electrically  driven,  with  two  recep- 
tacles for  tubes,  and  enclosed  in  a  safety  shield  (Fig.  162). 

Sterile  centrifuge  tubes  (10  c.c.  capacity),  Fig.  163. 


FIG.  162. — Small  electrical  centrifuge. 

Sterile  pipettes  (10  c.c.  graduated)  in  case. 

Sterile  glass  capsules  (in  case). 

Sterile  test-tubes.  IG' 


Sterile  all  glass  syringe  (5  c.c.  or  10  c.c.  capa- 
city) and  needle. 
Reagents  Required: 

Normal  saline  solution. 

10  per  cent,  sodium  citrate  solution  in  normal  saline. 

Human  blood  (vide  infra}. 

METHOD. — 

1.  Select  a  healthy  full-grown  rabbit  of  not  less  than 
2500  grammes  weight  in  accordance  with  the  directions 
already  given  (page  322)  and  prepare  it  for  intraperi- 
toneal  inoculation. 

2.  Measure  out  2  c.c.  citrated  human  blood  (collected 
at  a  surgical  operation  or  a  venesection,  or  withdrawn 
by  venipuncture  from  the  median  basilic  or  median 
cephalic  vein  of  a  normal  adult)  into  a  centrifuge  tube 
and  centrifugalise  thoroughly. 


328  METHODS    OF   IDENTIFICATION   AND    STUDY 

3.  Wash   with  three   changes  of  normal  saline  (vide 
also  page  388). 

4.  Transfer  the  washed  cells  to  a  sterile  capsule  by 
means  of  a  sterile  pipette.     Add  5  c.c.  of  normal  saline 
and  mix  thoroughly. 

5.  Take  up  the  mixture  of  cells  and  saline  in  the  all- 
glass  syringe  and  inject  into  the  peritoneal  cavity  of  the 
rabbit. 

6.  Seven    days   later    inject    intraperitoneally    the 
washed  cells  from  5  c.c.  human  blood  mixed  with  5  c.c. 
normal  saline. 

7.  Seven  days  later  inject  the  washed  cells  from  10 
c.c.  human  blood  mixed  with  5  c.c.  normal  saline. 

8.  After  a  further  interval  of  seven  days  repeat  the 
injection  of  washed  cells  from   10  c.c.  human  blood 
mixed  with  5  c.c.  normal  saline. 

NOTE. — Better  results  are  obtained  if  the  second  and  subsequent 
injections  are  made  intravenously,  even  when  smaller  quantities 
of  washed  red  cells  are  employed.  If,  however,  the  intravenous 
route  is  selected  exceeding  great  care  must  be  exercised  to  avoid 
the  introduction  of  air  into  the  vein — an  accident  which  is  fol- 
lowed, within  a  few  minutes,  by  the  death  of  the  rabbit  from 
pulmonary  embolism. 

9.  Allow  five  days  to  elapse,  then  collect  a  prelimi- 
nary sample  of  blood,  say  about  2  c.c.,  from  the  rabbit's 
ear.     Allow  it  to  clot,  separate  off  the  serum  and  trans- 
fer  to   a   sterile   test-tube.     Place  the  test-tube  in  a 
water-bath  at  56°  C.  for  fifteen  minutes  (to  inactivate) 
and  test  the  serum  quantitatively  for  haemolytic  prop- 
erties in  the  following  manner: 

THE  TITRATION  OF  HAEMOLYTIC  SERUM. 

Apparatus  Required, 
Electrical  centrifuge. 
Sterile  centrifuge  tubes. 
Water-bath  regulated  at  56°  C. 
Sterilised  pipettes  10  c.c.  graduated  in  tenths. 
Sterilised  pipettes  i  c.c.  graduated  in  tenths. 


TITRATING   H^MOLYSIN  329 

Sterile  test-tubes,  16X2  cm. 
Small  sterile  test-tubes,  9X1  cm. 
Small  test-tube  rack,  or  roll  of  plasticine. 
Capillary  teat  pipettes. 

Stout  rubber  band  or  length  of  small  rubber  tubing. 
Reagents  Required  and  Method  of  Preparation: 

1.  Normal  saline  solution. 

2.  Haemolytic    serum    inactivated    by   preliminary  heating  to 
56°  C.  for  15  minutes  (vide  supra}  in  test-tube  labelled  H.  S. 

3.  Complement.     Fresh  guinea-pig  serum  in  test-tube  labelled  C. 

Kill  a  normal  guinea-pig  with  chloroform  vapour. 

Open  the  thorax  with  all  aseptic  precautions,  and  collect 

as  much  blood  as  possible  from  the  heart  with  a  sterile 

Pasteur  pipette. 
Transfer  it  to  a  sterile  centrifuge  tube  and  place  the  tube 

in  the  incubator  at  37°  C.     Two  hours    later    separate 

the  clot  from  the  sides  of  the  tube,   and   centrifugalise 

thoroughly. 
Pipette  off  the  clear  serum  to  a  clean  sterilised  test-tube. 

4.  Erythrocyte  solution,  in  test-tube  labelled  E. 

Collect  and  wash  human  red  blood  cells  (see  page  388,  1-8). 
Measure  the  volume  of  red  cells  available  and  prepare  a  2 
per  cent,  suspension  in  normal  saline  solution. 

METHOD. — 

1.  Take  two  test-tubes  and  number  them  i  and  2, 
and  pipette  into  each  9  c.c.  of  normal  saline  solution. 

2.  Add  i  c.c.  of  haemolytic  rabbit  serum  to  tube  No.  i 
and  mix  thoroughly :  take  up  i  c.c.  of  the  mixture  and 
add  it  to  tube  No.  2 ;  mix  thoroughly. 

3.  Set  up  ten  small  test-tubes  in  test-tube  rack  or  in 
roll  of  plasticine,  and  number  i  to  10. 


Pipette   into   tube     No.     i    0.5    c.c.  =0.5    c.c. 

hagmolytic  serum 
Pipette  into  tube  No.  2  o.i  c.c.  =  0.1  c.c. 

haemolytic  serum 

Pipette    into    tube    No.   3    0.5  c.c.  =  0.05    c.c. 

haemolytic  serum 
Pipette  into  tube  No.  4  0.3  c.c.  =0.03  c.c. 

haemolytic  serum 
Pipette  into  tube  No.  5  0.2  c.c.  =0.02  c.c. 

haemolytic  serum 
Pipette  into  tube  No.  6  o.i  c.c.  =  0.01  c.c. 

haemolytic  serum 


From  tube 
H.  S. 


From 
tube  i. 


330  METHODS    OF   IDENTIFICATION   AND    STUDY 

Pipette  into  tube    No.  7    0.5    c.c.  =  0.005   c-c- 

haemolytic  serum 
Pipette   into   tube    No.  8    0.3     c.c.  =0.003  c.c. 

haemolytic  serum  From 

Pipette  into  tube    No.  9     0.2     c.c.  =0.002  c.c.    f     tube  2. 

haemolytic  serum 
Pipette  into  tube  No.  10    o.i     c.c.  =0.001  c.c. 

haemolytic  serum 

5.  To  each  tube  add  i  c.c.  of  erythrocyte  solution. 

6.  When  necessary  (that  is  to  say  in  tubes  2,  4,  5,  6, 
8,  9  and  10)  add  normal  saline  solution  to  the  mixture 
in  the  test-tubes  till  the  column  of  fluid  in  each  reaches 
to  the  same  level. 

7.  Shake  each  tube  in  turn,  so  as  to  thoroughly  mix 
its  contents.     Plug  the  mouth  of  each  tube  with  cotton 
wool,  and  place  entire  set  in  the  incubator  at  37°  C. 
for  one  hour. 

8.  Remove  the  tubes  from  the  incubator  and  into 
each  tube  pipette  o.i   c.c.   complement    (guinea-pig's 
serum)  and  replace  tubes  in  incubator  at   37°  C.  for 
further  period  of  one  hour. 

9.  Remove  the  tubes  from  the  incubator,  and  if  com- 
plete haemolysis  has  not  taken  place  in  every  tube, 
stand  on  one  side,  preferably  in  the  ice  chest,  for  an 
hour. 

10.  Then  examine  the  tubes. 

Complete  haemolysis  is  indicated  by  a  clear  red 
solution,  with  no  deposit  of  red  cells  at  the 
bottom  of  the  test-tube. 

Absence  of  haemolysis  is  indicated  by  a  clear  or 
turbid  colourless  fluid,  with  a  deposit  of  red 
cells  at  the  bottom  of  the  test-tubes. 

The  smallest  amount  of  haemolytic  serum  that  has 
caused  complete  haemolysis  is  known  as  the  minimal 
haemolytic  dose  (M.  H.  D.)  and  if  haemolysis  has 
occurred  in  all  the  tubes  down  to  No.  7 — the  m.  h.  d. 
of  this  particular  serum  is  .005  c.c.  =  200  minimal 


STORAGE  OF  H^EMOLYSIN  33! 

haemolytic  doses  per  cubic  centimetre.  Such  a  serum 
is  strong  enough  for  experimental  work;  indeed,  for 
many  purposes,  complete  haemolysis  down  to  tube  6 
will  indicate  a  serum  sufficiently  strong  (  =  100  m.  h.  d. 
per  cubic  centimetre).  If,  however,  only  the  first  one 
or  two  tubes  are  completely  haemolysed,  this  is  an 
indication  that  the  rabbit  should  receive  further  in- 
jections in  order  to  raise  the  haemolytic  power  to  a 
sufficiently  high  level. 

STORAGE  OF  H^EMOLYSIN. 

If,  and  when  the  haemolysin  content  of  the  rabbit's 
serum  is  found  to  be  sufficient,  destroy  the  animal  by 
chloroform  vapour. 

Remove  as  much  of  its  blood  as  possible  from  the 
heart  under  aseptic  precautions  into  sterilized  centri- 
fuge tubes. 

Transfer  the  tubes  of  blood  to  the  incubator  at 
37°  C.  for  two  hours — then  centrifugalize  thoroughly. 

Pipette  off  the  clear  serum,  and  fill  in  quantities  of 
i  c.c.,  into  small  glass  ampoules  or  pipettes,  and  her- 
metically seal  in  the  blow-pipe  flame,  care  being  taken 
to  avoid  scorching  the  serum. 

Place  the  ampoules  when  filled  with  serum  and  sealed, 
in  a  water-bath  at  56°  C.  for  30  minutes.  This  destroys 
the  complement,  i.  e.,  inactivates  the  serum,  and  at  the 
same  time,  provided  the  various  operations  have  been 
carried  out  under  aseptic  precautions,  ensures  its 
sterility.  A  longer  exposure  reduces  the  haemolytic 
power. 

Place  the  ampoules  in  a  closed  metal  box  and  store 
in  the  ice  chest  for  future  use. 


XVII.    EXPERIMENTAL  INOCULATION  OF 
ANIMALS. 

The  use  of  living  animals  for  inoculation  experiments 
may  become  a  necessary  procedure  in  the  Bacterio- 
logical Laboratory  for  some  one  or  more  of  the  follow- 
ing reasons : 

A.  Determination  of  Pathogenetic  Properties  of  Bac= 
teria  already  Isolated  in  Pure  Culture  (see  page  315). 

The  exact  study  of  the  conditions  influencing  the 
virulence  (including  its  maintenance,  exaltation  and 
attenuation)  of  an  organism,  and  precise  observations 
upon  the  pathogenic  effects  produced  by  its  entrance 
into,  and  multiplication  within  the  body  tissues  can 
obviously  only  be  carried  out  by  means  of  experimental 
inoculation;  whilst  many  points  relating  to  vitality, 
longevity,  etc.,  can  be  most  readily  elucidated  by  such 
experiments. 

B.  Isolation  of  Pathogenetic  Bacteria. 

Certain  highly  parasitic  bacteria  (which  grow  with 
difficulty  upon  the  artificial  media  of  the  laboratory) 
can  only  be  isolated  with  considerable  difficulty  from 
associated  saprophytic  bacteria  when  cultural  methods 
alone  are  employed ;  but  if  the  mixture  of  parasite  and 
saprophytes  is  injected  into  an  animal  susceptible  to 
the  action  of  the  former,  the  pathogenic  organism  can 
readily  be  isolated  from  the  tissues  of  the  infected 
animal.  The  pneumococcus  for  example  occurs  in 
the  sputum  of  patients  suffering  from  acute  lobar 
pneumonia,  but  usually  in  association  with  various 
saprophytes  derived  from  the  mouth  and  pharynx. 
The  optimum  medium  for  the  growth  of  the  pneumo- 
coccus, blood  agar,  is  also  an  excellent  pabulum  for  the 

332 


STUDY    OF    THE    PROBLEMS    OF    IMMUNITY  333 

saprophytes  of  the  mouth,  and  plate  cultures  are 
rapidly  overgrown  by  them  to  the  destruction  of  the 
more  delicate  pneumococcus.  But  inoculate  some  of 
the  sputum  under  the  skin  of  a  mouse  and  three  or 
four  days  later  the  pneumococcus  will  have  entered 
the  blood  stream  (leaving  the  saprophytes  at  the  seat 
of  inoculation)  and  killed  the  animal.  Cultivations 
made  at  the  post-mortem  (see  page  398)  from  the 
mouse's  heart  blood  will  yield  a  pure  growth  of  the 
pneumococcus. 

C.  Identification  of  Pathogenetic  Bacteria. 

The  resemblances,  morphological  and  cultural,  exist- 
ing between  certain  pathogenetic  bacteria  are  in  some 
cases  so  great  as  to  completely  overwhelm  the  differ- 
ences; again  the  same  bacterium  may  under  varying 
conditions  assume  appearances  so  different  from  those 
regarded  as  typical  or  normal  as  to  throw  doubt  on  its 
identity.  In  each  case  a  simple  inoculation  experiment 
may  decide  the  point  at  once.  As  a  concrete  example 
may  be  instanced  an  autopsy  on  an  animal  dead  from 
an  unknown  infection.  Cultivations  from  the  heart 
blood  gave  a  pure  growth  of  a  typical  (capsulated) 
pneumococcus.  Cultivations  from  the  liver  gave  a 
pure  growth  of  what  appeared  to  be  a  typical  (non- 
capsulated)  Streptococcus  pyogenes  longus.  The  latter 
inoculated  into  a  rabbit  caused  the  death  of  the  animal 
from  pneumococcic  septicaemia,  and  cultures  from  the 
rabbit's  blood  gave  a  pure  growth  of  a  typical  (capsu- 
lated) pneumococcus. 

D.  Study  of  the  Problems  of  Immunity. 

It  is  only  by  a  careful  and  elaborate  study  of  the 
behaviour  of  the  animal  cell  and  the  body  fluids  vis-a- 
vis with  the  infecting  bacterium  that  it  becomes  pos- 
sible to  throw  light  upon  the  complex  problem  whereby 
the  cell  opposes  successful  resistance  to  the  diffusion 
of  the  invading  microbe,  or  succeeds  in  driving  out 


334  EXPERIMENTAL   INOCULATION    OF  ANIMALS 

the  microbe   subsequently  to  the  occurrence  of  that 
diffusion. 

At  the  moment,  however,  our  attention  is  directed 
to  the  first  of  these  broad  headings,  for  it  is  by  the 
application  of  the  knowledge  acquired  in  its  pursuit 
that  we  are  able  to  deal  with  problems  arising  under 
any  of  the  remainder. 

For  whatever  purpose  the  inoculation  is  performed, 
it  is  essential  that  the  experiment  should  be  planned  to 
secure  the  maximum  amount  of  information  and  the 
minimum  of  discomfort  to  the  animal  used.  Every 
care  therefore  must  be  taken  to  ensure  that  the  virus  is 
introduced  into  the  exact  tissue  or  organ  selected ;  and 
the  operation  itself  must  be  carried  out  with  skill  and 
expedition,  and  under  strictly  aseptic  conditions. 

In  the  course  of  inoculation  studies  many  instances  of 
natural  immunity,  both  racial  and  individual,  will  be 
met  with;  but  it  must  be  recollected  that  natural  im- 
munity is  relative  only  and  never  absolute,  and  care 
be  taken  not  to  label  an  organism  as  non-pathogenic 
until  many  different  methods  of  inoculation  have  been 
performed  upon  different  species  of  animals,  combined 
when  necessary  with  various  procedures  calculated  to 
overcome  any  apparent  immunity,  and  have  invariably 
given  negative  results. 

In  some  countries  experiments  upon  animals  are 
only  permitted  under  direct  license  from  the  Govern- 
ment, and  then  only  within  premises  specially  licensed 
for  the  purpose.  In  England  this  license  is  in  the 
grant  of  the  Home  Secretary,  and  confers  the  permis- 
sion to  experiment  upon  animals  under  general  anaes- 
thesia, provided  that  after  the  experiment  is  completed 
the  animal  must  be  destroyed  before  regaining  con- 
sciousness. If  it  is  intended  to  carry  out  simple 
hypodermic  inoculations  and  superficial  venesections, 
Certificate  A,  granting  this  specific  permission  and 
dispensing  with  the  necessity  for  general  anaesthesia 


PREPARATION  335 

must  be  obtained  in  addition  to  the  license;  whilst  if 
the  inoculation  entails  more  extensive  operative  pro- 
cedures, and  it  is  necessary  to  observe  the  subsequent 
course  of  the  infection,  should  such  occur,  the  license 
must  be  coupled  with  Certificate  B — since  this  certifi- 
cate removes  the  compulsion  to  destroy  the  animal 
whilst  under  the  anaesthetic.  Further  special  certifi- 
cates and  combinations  of  certificates  are  required  if 
cats,  dogs,  horses,  asses  or  cattle  are  to  be  the  subjects 
of  experiment.  Under  every  certificate  it  is  expressly 
stipulated  that  if  the  animal  shows  signs  of  pain  it  must 
be  destroyed  immediately. 

The  animals  generally  employed  in  the  study  of  the 
pathogenic  properties  of  the  various  micro-organisms 
are: 

Hot  Blooded. 
Fowl. 
Pigeon. 


Cold  Blooded 

Warm  Blooded. 

Frog. 
Toad. 

Mouse. 
Rat. 

Lizard. 

Guinea  pig. 
Rabbit. 

Monkey. 

Preparation. — Before  inoculation,  the  experimental 
animals  should  be  carefully  examined,  to  avoid  the 
risk  of  employing  such  as  are  already  diseased:  since 
it  must  be  remembered  that  in  a  state  of  nature,  as 
well  as  in  captivity,  the  animals  employed  for  labora- 
tory inoculations  are  subject  to  infection  by  various 
animal  and  vegetable  parasites,  and  in  some  instances 
such  infection  presents  no  symptoms  which  are  ob- 
vious to  the  casual  examination;  the  sex  should  be 
noted,  the  weight  recorded,  and  the  rectal  tempera- 
ture taken.  The  remaining  items  of  importance  are 
the  time  of  the  inoculation,  the  material  that  is  inocu- 
lated, and  the  method  of  inoculation,  and  finally  under 
what  authority  the  experiment  is  performed.  In  the 
author's  laboratory  these  data  are  entered  upon  a  pink 
card  which  forms  part  of  a  card  index  system.  The 


336 


EXPERIMENTAL   INOCULATION    OF   ANIMALS 


card  further  provides  space  for  notes  on  the  course  of 
the  resulting  infection,  and  carries  on  the  reverse  the 
weight  and  temperature  chart  (Figs.  164  and  165). 

Preliminary  Inspection  and  Examination. — The  pre- 
liminary   examination    should    comprise    observation 


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THE     RABBIT 


337 


and  abdomen;  and  in  many  cases  the  microscopical 
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touching  upon  the  various  fleas,  lice  and  ticks  which 
at  times  infect  the  ordinary  laboratory  animals. 

The  Rabbit,  particularly  in  captivity,  is  subject  to 


338  EXPERIMENTAL    INOCULATION    OF  ANIMALS 

attacks  of  Psoric  Acari,  and  the  infection  is  readily 
transmitted  to  rabbits  in  neighbouring  cages  and  also 
to  guinea  pigs,  but  not  to  rats  and  mice.  One  species 
(Sarcoptes  minor  var.  cuniculi)  gives  rise  to  the  ordi- 
nary mange.  The  infection  first  shows  itself  as  thick 
yellowish  scales  and  crusts  around  the  nose,  mouth 
and  eyes,  spreads  to  the  bases  and  outer  surfaces  of 
the  ears  (never  to  the  inside  of  the  concha),  to  the 
fore  and  hind  legs  and  into  the  groins  and  around 
the  genitals.  The  acari  can  be  readily  demonstrated 
microscopically  in  scrapings  of  the  skin,  treated  with 
liquor  potassae.  Another  form  of  scabies  (due  to 
Psoroptes  communis  cuniculi)  commences  at  the  bottom 
of  the  concha,  which  is  filled  with  whitish-yellow  masses 
consisting  of  dried  crusts,  scales,  faeces,  and  dead  acari. 
The  base  of  the  ear  is  hard  and  swollen,  and  lifting  the 
animal  by  the  ears — as  is  usually  done — gives  rise  to 
considerable  pain;  indeed  this  symptom  may  be  the 
one  which  first  attracts  attention  to  an  infection,  which 
causes  progressive  wasting  and  terminates  in  death. 
A  mixed  infection — sarcoptic  plus  psorotic  acariasis — 
is  sometimes  seen. 

If  it  is  decided  to  try  and  save  animals  suffering  from 
infection  by  these  parasites,  they  must  be  segregated, 
the  scabs  carefully  cleaned  from  the  infected  areas  and 
the  denuded  surfaces  washed  with  5  per  cent,  solution 
of  Potassium  persulphate  (a  few  drops  being  allowed 
to  run  into  the  concha) ,  or  with  a  preparation  contain- 
ing equal  parts  of  soft  paraffin  and  vaseline  with  a  few 
drops  of  lysol.  This  treatment  should  be  repeated 
daily  until  the  acarus  is  destroyed  and  the  animal 
has  regained  its  normal  condition.  The  cages  should 
be  disinfected  and  all  neighbouring  animals  carefully 
examined,  and  any  which  show  signs  of  infection 
should  be  treated  in  a  similar  manner.  Favus  also 
attacks  the  rabbit,  and  the  typical  spots  are  first  noted 
around  the  base  of  the  ear. 


THE    MONKEY  339 

Infection  by  Coccidium  oviforme  is  very  common, 
without  however  presenting  any  symptoms  by  which 
the  infection  may  be  recognised.  Usually  the  condi- 
tion is  only  noted  post-mortem,  when  the  liver  is  found 
to  be  studded  with  numerous  caseating  tubercles, 
which  on  examination  prove  to  be  cystic  areas  crowded 
with  coccidia.  Sometimes  too  the  liver  of  a  rabbit 
dead  from  some  intentional  or  accidental  bacterial 
infection  is  found  at  the  post-mortem  to  be  marked  by 
fine  yellowish  streaks  and  small  tubercles  due  to  the 
embryos  of  Tcznia  serrdta,  while  the  cystic  form 
(Cysticercus  pisiformis)  is  often  noted  free  in  the 
peritoneal  cavity,  or  invading  the  mesentery. 

Abscess  formation  from  infection  with  ordinary 
pyogenic  bacteria  occurs  naturally  in  the  rabbit,  and  fre- 
quently the  animal  house  of  a  laboratory  is  decimated 
by  an  infective  septicaemia  due  to  B.  cuniculicida. 

The  Mouse  and  Rat  suffer  from  septicaemia,  and  from 
the  cysticercus  form  of  T&nia  murina;  the  cystic  form 
(Cysticercus  fasciolaris)  of  T.  crassicollis  has  its  habitat 
in  their  livers.  These  small  rodents  are  frequently 
infected  with  scabies,  but  if  freely  provided  with  clean 
straw  will  clean  themselves  by  rubbing  through  it. 
The  mouse  is  also  attacked  by  favus,  and  the  rat  is 
often  infected  with  Trypanosoma  Lewisi. 

The  Guinea  pig,  like  the  rabbit,  suffers  from  scabies 
and  coccidiosis.  In  addition  it  is  often  naturally 
infected  with  B.  tuberculosis,  and  it  is  a  wise  precaution 
to  test  animals  as  soon  as  they  reach  the  laboratory  by 
injecting  Koch's  Old  Tuberculin — 0.5  c.c.  causing  death 
in  the  tuberculous  cavy  within  48  hours. 

The  Monkey  is  naturally  prone  to  tuberculosis,  and 
should  be  injected  with  i  c.c.  Old  Tuberculin  on 
arrival  in  the  laboratory.  The  tissues  of  the  monkey 
also  serve  as  the  habitat  for  a  Nematode  worm  para- 
sitic in  cattle  (CEsophagostoma  inflatum)  resembling  the 
Anchylostomum,  and  this  parasite  frequently  bores 


340  EXPERIMENTAL   INOCULATION    OF  ANIMALS 

through  the  intestinal  wall,  and  provokes  the  forma- 
tion of  small  cysts  in  the  immediately  adjacent  mesen- 
tery. The  presence  of  these  cysts  may  give  rise  to 
considerable  speculation  at  the  post-mortem. 

The  Pigeon  may  be  infected  by  Hczmosporidia,  and 
its  blood  show  the  presence  of  halteridia.  This  bird 
may  also  be  the  subject  of  a  bacterial  infection  known 
as  pigeon  diphtheria;  while  the  fowl  may  be  subject 
to  scabies  and  ringworm,  or  suffer  from  fowl  cholera 
or  fowl  septicaemia — infections  due  to  members  of 
the  haemorrhagic  septicaemia  group. 

Weighing. — The  larger  animals  are  most  conveniently 
weighed  in  a  decimal  scale  provided  with  a  metal  cage 
for  their  reception  instead  of  the  ordinary  pan  (Fig. 
1 66).  Mice  and  rats  are  weighed  in  a  modification 


FIG.  166. — Rabbit  scales. 

of  the  letter  balance,  weighing  to  250  grammes,  which 
has  a  conical  wire  cage,  (carefully  counterpoised) 
substituted  for  its  original  pan  (Fig.  167) . 

Temperature. — To  take  the  rectal  temperature  of 
any  of  the  laboratory  animals,  the  animal  should  be 
carefully  and  firmly  held  by  an  assistant.  Introduce 
the  bulb  of  an  ordinary  clinical  thermometer,  well 
greased  with  vaseline,  just  within  the  sphincter  ani. 
Allow  it  to  remain  in  this  position  for  a  few  seconds, 


CAGES 


341 


and  then  push  it  on  gently  and  steadily  until  the  en- 
tire bulb  and  part  of  the  stem,  as  far  as  the  constric- 
tion, have  passed  into  the  rectum.  Three  to  five  min- 
utes later,  the  time  varying  of  course  with  the  sensi- 
bility of  the  thermometer  used,  withdraw  the  instru- 
ment and  take  the  reading.  The  thermometers  em- 


FIG.  167. — Mouse  scales 

ployed  for  recording  temperature  should  be  verified 
from  time  to  time  by  comparision  with  a  standard 
Kew  certified  Thermometer  kept  in  the  laboratory 
for  that  purpose. 

Cages. — During  the  period  which  elapses  between 
inoculation  and  death,  or  complete  recovery,  the  ex- 
perimental animals  must  be  kept  in  suitable  recep- 
tacles which  can  easily  be  kept  clean  and  readily 
disinfected. 

The  mouse  is  usually  stored  in  a  glass  jar  (Fig.  168) 


342 


EXPERIMENTAL   INOCULATION    OF   ANIMALS 


ii  cm.  high  and  n  cm.  in  diameter,  closed  by  a  wire 
gauze  cover  which  is  weighted  with  lead  or  fastened 
to  the  mouth  of  the  jar  by  a  bayonet  catch.  A  small 
oblong  label,  5  cm.  by  2.5  cm.,  sand-blasted  on  the 
side  of  the  cylinder,  is  a  very  convenient  device  as 
notes  made  upon  this  with  an  ordinary  lead  pencil 
show  up  well  and  only  require  the  use  of  a  damp  cloth 
to  remove  them  (Fig.  168). 

The  rat  is  kept  under  observation  in  a  glass  jar  simi- 
lar, but  larger,  to  that  used  for  the  mouse. 


FIG.  168. — Mouse  jar. 


FIG.  169.— Tripod. 


A  layer  of  sawdust  at  the  bottom  of  the  jar  absorbs 
any  moisture  and  cotton- wool  or  paper  shavings  should 
be  provided  for  bedding.  The  food  should  consist  of 
bran  and  oats  with  an  occasional  feed  of  bread-and- 
milk  sop. 

The  use  of  a  metal  tripod,  on  the  platform  of  which 
are  soldered  two  small  cups  for  the  reception  of  the 
food,  inside  the  cage,  prevents  waste  of  food  or  its  con- 
tamination with  excreta  (Fig.  169). 

After  use  the  jars  and  tripods  are  sterilised  either  by 
chemical  reagents  or  by  autoclaving. 

The  rabbit  and  the  guinea-pig  are  confined  in  cages  of 
suitable  size,  made  entirely  of  metal  (Fig.  170).  The 


CAGES 


343 


sides  and  top  and  bottom  are  of  woven  wire  work; 
beneath  the  cage  is  a  movable  metal  tray  filled  with  saw- 
dust, for  the  reception  of  the  excreta.  The  cage  as  a 
whole  is  raised  from  the  ground  on  short  legs .  The  sides, 
etc.,  are  generally  hinged  so  that  the  cage  packs  up 
flat,  for  convenience  of  storing  and  also  of  sterilising. 

The  ordinary  rat  cage,  a  rectangular  wire- work  box, 
30  cm.  from  front  to  back,  20  cm.  wide,  and  14  cm. 
high,  makes  an  excellent  cage  for  guinea-pigs  if  fitted 
with  a  shallow  zinc  tray,  35  cm.  by  24  cm.,  for  it  to 
stand  upon. 


FIG,  170. — Metal  rabbit  rage. 

A  plentiful  supply  of  straw  should  be  provided 
for  bedding  and  the  food  should  consist  of  fresh  vege- 
tables, cabbage  leaves,  carrot  and  turnip  tops  and  the 
like  for  the  morning  meal  and  broken  animal  biscuits 
for  the  evening  meal.  Occasionally  a  little  water  may 
be  placed  in  the  cage  in  an  earthenware  dish. 

The  tray  which  receives  the  dejecta  should  be 
cleaned  out  and  supplied  with  fresh  sawdust  each  day, 
and  the  soiled  sawdust,  remains  of  food,  etc.,  should 
be  cremated. 

These  cages  are  sterilised  after  use  either  by  auto- 
claving  or  spraying  with  formalin. 


344  EXPERIMENTAL   INOCULATION    OF  ANIMALS 

As  animal  inoculation  is  purely  a  surgical  operation, 
the  necessary  instruments  will  be  similar  to  those  em- 
ployed by  the  surgeon,  and,  like  them,  must  be  sterile. 
In  the  performance  of  the  inoculation  strict  attention 
must  be  paid  to  asepsis,  and  suitable  precautions 
adopted  to  guard  against  accidental  contamination  of 
the  material  to  be  introduced  into  the  animal.  In 
addition,  the  hands  of  the  operator  should  be  care- 
fully disinfected. 

The  list  of  apparatus  used  in  animal  inoculations 
given  below  comprises  practically  everything  needed 
for  any  inoculation.  Needless  to  remark,  all  the  appa- 
ratus will  never  be  required  for  any  one  inoculation. 

Apparatus  Required  for  Animal  Inoculation : 

i.  Water  steriliser  (vide  page  33) .  It  is  also  convenient  to  have 
a  second  water  steriliser,  similar  but  smaller  (23  by  7  by  5  cm.), 
for  the  sterilisation  of  the  syringes. 


fii  i>-iiv 

LIpPP 

\ 

FIG.  171. — Hypodermic  syringe  with  ringer  rests. 

2.  Injection  syringe.     The  best  form  is  one  of  the  ordinary 
hypodermic  pattern,  i  c.c.  capacity  graduated  in  twentieths  of  a 
cubic  centimeter  (0.05  c.c.),  fitted  with  finger  rests,  but  with  the 
leather  washers  and  the  packing  of  the  piston  replaced  by  those 
made  of  asbestos  (Fig.  171).     The  instrument  must  be  easily  taken 
to  pieces,  and  spare  parts  should  be  kept  on  hand  to  replace  acci- 
dental breakage  or  loss.     Other  useful  syringes  are  those  of  2  c.c., 
5  c.c.,  10  c.c.,  and  20  c.c.  capacity.     A  good  supply  of  needles  must 
be  kept  on  hand,  both  sharp-pointed  and  with  blunt  ends.     To 
sterilise  the  syringe,  fill  it  with  water,  loosen  the  packing  of  the 
piston  and  all  the  screw  joints,  place  it  in  the  steriliser  and  boil 
for  at  least  five  minutes.     Disinfect  the  syringe  after  use,  in  a 
similar  manner.     The  needles,  which  are  exceedingly  apt  to  rust 
after  being  boiled,  should  be  stored  in  a  pot  of  absolute  alcohol 
when  not  in  use. 

3.  Operating  table. 

4.  Surgical  instruments.     Sterilise  these  before  use  by  boiling, 
and  disinfect  them  after  use  by  the  same  means.     Wipe  perfectly 
dry  immediately  the  disinfection  is  completed. 


APPARATUS    REQUIRED    FOR   ANIMAL   INOCULATION    345 

Scissors,  probe  and  sharp-pointed. 

Dissecting  forceps  of  various  patterns. 

Pressure  forceps. 

Retractors  (small  self  retaining  Fig.  172). 

Aneurism  needles,  sharp  and  blunt. 

Scalpels,         j 

Keratomes,    >    with  metal  handles. 

Trephines,     j 

Michel's  steel  clips  and  special  forceps  for  applying  the  same. 

These    small  steel   clips  enable  the  operator  to  easily  and 

rapidly  close  skin  incisions  and  are  most  satisfactory  for 

animal  operations. 
Surgical  needles. 
Needle  holder. 

Soft  rubber  catheters,  various  sizes. 
Gum  elastic  cesophageal  bougies  with  con-      FIG.  172.     Small 

nection  to  fit  syringe.  self   retaining   re- 

5.  Anesthetic. 

(a)  General:  The  safest  general  anaesthetic  for  animals  is  an 
A.  C.  E.  mixture,  freshly  prepared,  containing  by 
volume  alcohol  i  part,  chloroform  2  parts,  ether  6  parts, 
and  should  be  administered  on  a  "cone"  formed  by 
twisting  up  one  corner  of  a  towel  and  placing  a  wad  of 
cotton-wool  inside  it,  or  from  a  saturated  cotton-wool 
pad  packed  into  the  bottom  of  a  small  beaker. 

(6)    Local: 

1.  Cocaine  hydrochloride,  2  per  cent,  in  adrenalin  i  per 
mille  solution. 

2.  Beta-eucaine,    2   per  cent,  in  adrenalin,  i    per  mille 
solution. 

3.  Ethyl  chloride  jet. 

6.  Sterile  glass  capsules  of  various  sizes. 

10    c.c.    (in    tenths    of    a    cubic 

centimetre) . 

7.  Cases  of  sterile  pipettes  ,.     .        ,      , .,_       c          ,  . 

i  c.c.   (in  hundredths  of  a  cubic 

centimetre) . 

8.  Flasks  (75  c.c.)  containing  sterilised  normal  saline  solution 
(or  sterile  bouillon) . 

9.  Sterilised  cotton-wool.     Cotton-wool  (absorbent)  is  packed 
loosely  in  a  copper  cylinder  similar  to  that  used  for  storing  capsules, 
and  sterilised  in  the  hot-air  oven. 

10.  Sterilised  gauze.     Gauze  is  sterilised  in  the  same  way  as 
cotton-wool. 

11.  Sterilised  silk  and  catgut  for  sutures.     These  are  sterilised, 
as  required,  by  boiling  for  some  ten  minutes  in  the  water  steriliser. 

12.  Flexible  collodion  (or  compound  tincture  of  benzoin). 

13.  Grease  pencil. 

14.  Tie-on  celluloid  labels,  to  affix  to  the  cages. 


346  EXPERIMENTAL   INOCULATION    OF  ANIMALS 

15.  Razor. 

1 6.  Small  pot  of  warm  water. 

17.  Liquid  soap.     Liquid  soap  is  prepared  as  follows:  Meas- 
ure out  100  grammes  of  soft  soap  and  add  to  500  c.c.  of  2  per  cent. 
lysol  solution  in  a  large  glass  beaker;  dissolve  by  heating  in  a  water- 
bath  at  about  90°  C.      Bottle  and  label  "Liquid  Soap." 

1 8.  In  place  of  the  liquid  soap  and  razor  it  is  sometimes  con- 
venient to  use  a  Depilatory  powder. 

Barium  sulphide i  part 

Rice  starch 3  parts 

Dust  the  powder  thickly  over  the  aiea  to  be  denuded  of  hai.', 
sprinkle  with  water  and  mix  into  a  thin  paste  in  situ;  allow  the 
paste  to  act  for  three  minutes,  then  scrape  off  with  a  bone  spatula 
— the  hair  comes  away  with  the  paste  and  leaves  a  perfectly  bare 
patch.  This  process  is  preferably  carried  out,  the  day  previous  to 
the  operation. 

Material  Utilised  for  Inoculation. — The  material  in- 
oculated may  be  either — 

1.  Cultures  of  bacteria — grown  in  fluid  media,  or  on 
solid  media. 

2.  Metabolic  products  of  bacterial  activity — e.  g., 
toxins  in  solution. 

3.  Pathological  products  (fluid  secretions  and  excre- 
tions, solid  tissues). 

The  Preparation  of  the  Inoculum. — 
(a)   Cultivations  in  Fluid  Media. — 

1.  Flame  the  plug  of  the  culture  tube. 

2.  Remove  the  plug  and  flame  the  mouth  of  the 
tube. 

3.  Slightly  raise  the  lid  of  a  sterile  capsule,    insert 
the  mouth  of  the  culture  tube  into  the  aperture  and 
pour  some  of  the  cultivation  into  the  capsule. 

4.  Remove  the  mouth  of  the  culture  tube  from  the 
capsule,  replace  the  lid  of  the  latter,  flame  the  mouth 
of  the  tube,  and  replug. 

5.  Remove  the   syringe   from   the   steriliser,  squirt 
out  the  water  from  its  interior,  and  allow  to  cool. 

6.  Raise  the  lid  of  the  capsule  sufficiently  to  admit 
the  needle  of  the  syringe  and  draw  the  required  amount 
of  the  cultivation  into  the  barrel  of  the  syringe. 


MATERIAL    UTILISED    FOR    INOCULATION 


347 


(Or,  remove  a  definite  measured  quantity  of  the  cul- 
tivation directly  from  the  tube  or  flask  by  means  of 
a  sterile  graduated  pipette,  discharge  the  measured 
amount  into  a  sterile  capsule,  and  fill  into  the  syringe ; 
or  take  up  the  required  quantity  of  the  cultivation 
directly  into  the  graduated  syringe  from  the  tube  or 
flask. 

If  it  is  necessary  to  introduce  a  large  bulk  of  fluid 
into  the  animal,  the  cultivation  should  be  transferred 


FIG.  173. — Conical  separatory  funnel,  fitted  for  injection  of  fluid  cultivations. 

with  aseptic  precautions,  to  a  sterile  separatory  funnel, 
preferably  of  the  shape  shown  in  figure  173,  and  gradu- 
ated if  necessary.  This  is  supported  on  a  retort  stand 
and  raised  sufficiently  above  the  level  of  the  animal 
to  be  injected,  so  as  to  secure  a  good  "  fall."  A  piece  of 
sterilised  rubber  tubing  of  suitable  length,  fitted  with  an 
injection  needle  and  provided  with  a  screw  clamp,  is 
now  attached  to  the  nozzle  of  the  funnel  and  the  opera- 


348  EXPERIMENTAL   INOCULATION    OF  ANIMALS 

tion  completed  according  to  the  requirements  of  the 
particular  case. 

This  method  is  quite  satisfactory  when  the  injection 
is  made  into  the  pleural  or  abdominal  cavities  or  directly 
into  a  vein  but  if  the  injection  has  to  be  made  into  the 
subcutaneous  tissue  the  "fall"  may  not  be  sufficient  to 
force  the  fluid  in.  In  this  case  it  will  be  necessary  to 
transfer  the  culture  to  a  sterile  wash-bottle  and  fasten  a 
rubber  hand  bellows  to  the  air  inlet  tube  (interposing  an 
air  filter)  and  attach  the  tubing  with  the  injection 
needle  to  the  outlet  tube  (Fig.  174).  By  careful  use 
sufficient  force  can  be  obtained  to  drive  the  injec- 
tion in. 

(6)  Cultivations  on  Solid  Media  (e.  g.,  Sloped  Agar) . — 
i .   By  means  of  a  sterile  graduated  pipette  introduce 


FIG.  174. — Arrangement  of  pressure  injection  apparatus. 

a  suitable  small  quantity  of  sterile  bouillon  (or  sterile 
normal  saline  solution)  into  the  culture  tube. 

2.  With  a  sterile  platinum  loop  or  spatula  scrape 
the  bacterial  growth  off  the  surface  of  the  medium, 
and  emulsify  it  with  the  bouillon.     It  then  becomes 
to  all  intents  and  purposes  a  fluid  inoculum. 

3.  Pour  the  emulsion  into  a  sterile  capsule  and  fill 
the  syringe  therefrom. 


METHODS    OF    SECURING    ANIMALS  349 

(c)  Toxins. — Prepared      by     previously     described 
methods  (vide  page  318),  are  manipulated  in  a  similar 
manner  to  cultivations  in  fluid  media. 

(d)  Pathological  Products. — Fluid  secretions,  excre- 
tions, etc.,  such  as  serous  exudation,  pus,  blood,  etc., 
are  treated  as  fluid  cultivations ;  but  if  the  material  is 
very  thick  or  viscous,  a  small  quantity  of  sterile  bouillon 
or  normal  saline  solution  may  be  used  to  dilute  it,  and 
thorough  incorporation  effected  by  the  help  of  a  sterile 
platinum  rod. 

Solid  tissues,  such  as  spleen,  lymph  glands,  etc., 
may  be  divided  into  small  pieces  by  sterile  instruments 
and  rubbed  up  in  a  sterilised  agate  mortar  (using  an 
agate  pestle,  with  a  small  quantity  of  sterile  bouillon, 
and  the  syringe  filled  from  the  resulting  emulsion. 


FIG.  175. — Holding  rabbit  for  shaving. 

If  it  is  desired  to  inoculate  tissue  en  masse,  remove 
from  the  material  a  small  cube  of  i  or  2  mm.  and 
introduce  it  into  a  wound  made  by  sterile  instruments 
in  a  suitable  situation,  and  occlude  the  wound  by 
means  of  Michel's  steel  clips  and  a  sealed  dressing. 

Method  of  Securing  Animals  During  Inoculation. — 

For  the  majority  of  inoculations,  especially  when  no 
anaesthetic  is  administered,  it  is  customary  to  employ 
an  assistant  to  hold  the  animal  (see  Fig.  175). 

If  working  single  handed  Voge's  holder  for  guinea- 
pigs,  is  a  useful  piece  of  apparatus  the  method  of  using 
which  is  readily  seen  from  the  accompanying  figures 
(Figs.  176,  177). 

The  instrument  itself  consists  of  a  hollow  copper 


35° 


EXPERIMENTAL   INOCULATION    OF  ANIMALS 


cylinder,  one  end  of  which  is  turned  over  a  ring  of  stout 
copper  wire,  and  from  this  open  end  a  slot  is  cut  ex- 
tending about  half  way  along  one  side  of  the  cylinder. 
The  opposite  end  is  closed  by  a  " pull-off"  cap  and  is 
perforated  around  its  edge  by  a  row  of  ventilating  holes, 
which  correspond  'with  holes 
cut  in  the  rim  of  the  cap.  In 
the  event  of  the  animal  resist- 
ing attempts  to  remove  it  from 
the  holder  backwards,  this  cap 
is  taken  off  and  the  holder 
placed  on  the  table  and  the 
guinea-pig  allowed  to  walk  out. 

To  provide  for  different-sized 
animals,  two  sizes  of  this  holder 
will  be  found  useful : 

1.  Length,  16  cm.;  breadth,  6 
cm. ;  size  of  slot,  8  cm.  by  2.5  cm. 

2.  Length,    20  cm.;  breadth, 
8  cm.;  size  of  slot,    10  cm.  by 

2.5  cm. 

A  convenient  holder  for  mice  and  even  small  rats 
is  shown  in  figure  178,  the  tail  being  securely  held  by 
the  spring  clip.  Needless  to  say,  the  holder  should 


FiG.  176. — Taking  guinea- 
pig's  temperature. 


FIG.  177. — Voge's  holder. 

be  entirely  of  metal,  and  the  wire  cage  detachable  and 
easily  renewed. 

When  the  animal  is  anaesthetised,  it  is  more  conven- 
ient to  secure  it  firmly  to  some  simple  form  of  operat- 
ing table,  such  as  Tatin's  (Fig.  179),  which  will  accom- 


OPERATION    TABLE 


351 


modate  rabbits,  guinea-pigs,  and  rats:  or  to  the  more 
elaborate  table  devised  by  the  author  (Fig.  180). 

Operation  Table. — This  is  a  table  of  the  "aseptic" 
type,  composed  of  steel  tubing,  nickel-plated  or  enam- 
elled. The  table-top  frame  is  sufficiently  large  to 
accommodate  rabbits,  dogs  and  monkeys;  and  is  sup- 


FIG.  178. — Mouse  holder. 

ported  upon  telescopic  uprights,  so  that  it  is  adjustable 
as  to  height ;  in  its  long  axis  it  can  be  inclined  (at  either 
end)  to  45°  from  the  horizontal.  Further  it  can  be 
completely  rotated  about  its  long  axis.  The  table- 
top  itself  is  composed  of  a  sheet  of  copper  wire  gauze 


FIG.  179. — Taten's  operation  table. 

loosely  suspended  from  the  long  sides  of  the  tubular 
frame.  The  slackness  of  the  gauze  bed  permits  of  an 
india  rubber  hot  water  bottle,  or  an  electrotherm  being 
placed  under  the  animal,  and  if  during  the  course  of 
an  experiment  it  is  necessary  to  reverse  the  animal, 


352 


EXPERIMENTAL    INOCULATION    OF  ANIMALS 


the  table-top  frame  is  completely  rotated,  the  device 
adopted  for  suspending  the  gauze  is  detached  and  the 
gauze  reversed  also,  so  that  it  again  supports  the  animal 
from  below 


FIG.  1 80. — Author's  operating  table1. 
METHODS  OF  INOCULATION. 

The  following  methods  of  inoculation  apply  more 
particularly  to  the  rabbit,  but  from  them  it  will  readily 
be  seen  what  modifications  in  technique,  if  any,  are 
necessary  in  the  case  of  the  other  experimental  animals. 

1.  Cutaneous  Inoculation. — (Anesthetic,  none.) 

1.  Have  the  animal  firmly  held  by  an  assistant  (or 
secured  to  the  operating  table) . 

2.  Apply  the  liquid  soap  to  the  fur,  over  the  area 

1  This  table  is  made  by  Messrs.  Down  Bros.,  St.  Thomas's  Street,  London, 

o.    I,. 


SUBCUTANEOUS  353 

selected  for  inoculation,  with  a  wad  of  cotton-wool, 
and  lather  freely  by  the  aid  of  warm  water;  shave 
carefully  and  thoroughly;  or  apply  the  depilatory 
powder. 

3 .  Wash  the  denuded  area  of  skin  thoroughly  with 
2  per  cent,  lysol  solution. 

4.  Wash  off  the  lysol  with  ether  and  allow  the  latter 
to  evaporate. 

5.  Make  numerous  short,  parallel,  superficial  inci- 
sions with  the  point  of  a  sterile  scalpel. 

6.  When  the  oozing  from  the  incisions  has  ceased, 
rub  the  inoculum  into  the  scarifications  by  means  of 
the  flat  of  a  scalpel  blade,  or  a  sterile  platinum  spatula. 

7.  Cover  the  inoculated  area  with  a  pad  of  sterile 
gauze  secured  in  situ  by  strips  of  adhesive  plaster  or 
by  sealing  down  the  edges  of  the  gauze  with  collodion. 

8.  Release  the  animal,  place  it  in  its  cage,  and  affix 
a  label  upon  which  is  written : 

(a)  Distinctive  name  or  number  of  the  animal. 

(b)  Its  weight. 

(c)  Particulars  as  to  source  and  dose  of  inoculum. 

(d)  Date  of  inoculation. 

2.  Subcutaneous  Inoculation. — 

(a)  Fluid  Inoculum. — (Ancesthetic,  none.) 
Steps  1-4.     As  for  cutaneous  inoculation. 

5.  Pinch  up  a  fold  of  skin  between  the  forefinger 
and  thumb  of  the  left  hand;  take  the  charged  hypo- 
dermic syringe  in  the  right  hand,  enter  the  needle  into 
a  ridge  of  skin  raised  by  the  left  finger  and  thumb, 
and  push  it  steadily  onward  until  about  2  cm.  of  the 
needle  are  lying  in   the   subcutaneous   tissue.     Now 
release  the  grasp  of  the  left  hand  and  slowly  inject  the 
fluid  contained  in  the  syringe. 

6.  Withdraw  the  needle,  and  at  the  same  moment 
close  the  puncture  with  a  wad  of  cotton  wool,  to  prevent 
the  escape  of  any  of  the  inoculum.     The  injected  fluid, 

23 


354  EXPERIMENTAL   INOCULATION    OF   ANIMALS 

unless  large  in  amount,  will  be  absorbed  within  a  very 
short  time. 

7.  Label,  etc. 

(6)  Solid  Inoculum. — (Anesthetic,  none;  or  Ethyl 
chloride  spray.) 

Steps  1-4.     As  for  cutaneous  inoculation. 

5.  Raise  a  small  fold  of  skin  in  a  pair  of  forceps, 
and  make  a  small  incision  through  the  skin  with  a 
pair  of  sharp-pointed  scissors  or  with  the  point  of  a 
scalpel. 

6.  Insert  a  probe  through  the  opening  and  push  it 
steadily  onward  in  the  subcutaneous  tissue,  and  by 
lateral  movements  separate  the  skin  from  the  under- 
lying muscles  to  form  a  funnel-shaped  pocket  with  its 
apex  toward  the  point  of  entrance. 

7.  By  means  of  a  pair  of  fine-pointed  forceps  intro- 
duce a  small  piece  of  the  inoculum  into  this  pocket 
and   deposit  it  as  far  as  possible  from  the  point  of 
entrance. 


FIG.  181. — Glass  tube  syringe  for  subcutaneous  "solid"  inoculation. 

Or,  improvise  a  syringe  by  sliding  a  piece  of  glass 
rod  (to  serve  as  a  piston)  into  the  lumen  of  a  slightly 
shorter  length  of  glass  tubing  and  secure  in  position  by 
a  band  of  rubber  tubing.  Sterilise  by  boiling.  With- 
draw the  rod  a  few  millimetres  and  deposit  the  piece  of 
tissue  within  the  orifice  of  the  tube,  by  means  of  sterile 
forceps.  Now  pass  the  tube  into  the  depths  of  the 
"pocket,"  push  on  the  glass  rod  till  it  projects  beyond 
the  end  of  the  tube,  and  withdraw  the  apparatus, 
leaving  the  tissue  behind  in  the  wound. 

8.  Close  the  wound  in  the  skin  with  Michel's  clips 
and  a  dressing  of  gauze  sealed  with  collodion  (or  Tinct. 
benzoin) . 

9.  Label,  etc. 


INTRAPERITONEAL  355 

3.  Intramuscular.— 

(a)  Fluid  Inoculum. — (Anesthetic,  none.) 
Steps  1-4.     As  for  cutaneous  inoculation. 

5.  Steady   the   skin   over   the   selected   muscle   or 
muscles    with   the   slightly   separated   left    forefinger 
and  thumb. 

6.  Thrust  the  needle  of  the  injecting  syringe  boldly 
into   the   muscular   tissue   and   inject   the   inoculum 
slowly. 

7.  Label,  etc. 

(b)  Solid  Inoculum. — (Anesthetic,  A.  C.  E.) 

1.  Secure  the  animal  to  the  operation  table  and 
anaesthetise. 

2.  Shave  and  disinfect  the  skin  at  the  seat  of  opera- 
tion. 

3 .  Surround  the  field  of  operation  by  strips  of  gauze 
wrung  out  in  2  per  cent,  lysol  solution. 

4.  Incise  skin,  aponeurosis,  and  muscle  in  turn. 

5.  Deposit  the  inoculum  in  the  depths  of  the  incision. 

6.  Close  the  wound  in  the  muscle  with  buried  sutures 
and  the  cutaneous  wound  with  either  continuous  or 
interrupted  sutures  or  with  Michel's  steel  clips. 

7.  Apply  a  sealed  dressing  of  gauze  and  collodion. 

8.  Remove  the  animal  from  the  operating  table. 

9.  Label,  etc. 

4.  Intraperitoneal. — 

(a)  Fluid  Inoculum. — (Anesthetic,  none.) 

Steps  1-4.  As  for  cutaneous  inoculation.     Shave  a 

fairly   broad   transverse   area,    stretching   from   flank 

to  flank. 

5.  Place  the  left  forefinger  on  one  flank  and  the 
thumb  on  the  opposite,  and  pinch  up  the  entire  thick- 
ness of  the  abdominal  parietes  in  a  triangular  fold. 
Now,  by  slipping  the  peritoneal  surfaces  (which  are  in 
apposition)  one  over  the  other,  ascertain  that  no  coils 
of  intestine  are  included  in  the  fold. 


356  EXPERIMENTAL   INOCULATION    OF  ANIMALS 

6.  Take  the  syringe  in  the  right  hand  and  with  the 
needle  transfix  the  fold  near  its  base  (Fig.  182). 

7.  Now  release  the  fold,  but  hold  the  syringe  steady; 
as  the  parietes  flatten  out,  the  point  of  the  needle  is 
left  free  in  the  peritoneal  cavity  (see  Fig.  183). 


FIG.  182. — Intraperitoneal  inoculation — fluid. 

8.  Inject  the  fluid  from  the  syringe. 

9.  Label,  etc. 
Second  Method: 

Steps  1-4.     As  in  the  first  method. 

5.  Anaesthetise  a  small  selected 
area  of  skin  by  spraying  it  with 
ethyl  chloride. 

6.  Heat  platinum  searing  wire 
(0.5    mm.    wire,   twisted    to    the 

FIG.  183.— Section  of  ab-  shape    indicated    in    figure    184, 


dominal  wall,  etc.,  showing  -,   .                1                                 -,    ' 

point  of  needle  lying  free  mounted  in  an  aluminum  handle) 

in     the     peritoneal    cavity  to    redneSS,     and    with    it    bum    a 
above  the  coils  of  intestine. 


hole  through  the  anaesthetic  area 
of  skin  and  abdominal  muscle  down  to,  but  not  through, 
the  visceral  peritoneum. 

7.  Fix   a    blunt-ended    needle    on    to    the  charged 
syringe,  and  by  pressing  the  rounded  end  firmly  against 
the  peritoneum  it  can  easily  be  pushed  through  into  the 
peritoneal  cavity. 

8.  Inject  the  fluid  from  the  syringe. 


COLLODION    SACS  357 

9.  Label,  etc. 

This  method  is  especially  useful  when  it  is  desired 
to  collect  samples  of  the  peritoneal  fluid  from  time  to 
time  during  the  period  of  observation,  as  fluid  can  be 
removed  from  the  peritoneal  cavity,  at  intervals, 
through  this  aperture  in  the  abdominal  parietes,  by 
means  of  a  sterile  capillary  pipette. 


FIG.  184. — Platinum  wire  for  burning  hole  through  parietes. 

(b)  Solid  Inoculum  (or  the  implantation  of  capsules 
containing  fluid  cultivations). — (Anasthetic,  A.  C.  E.) 

1.  Anaesthetise   the   animal   and   secure   it   to   the 
operating  table. 

2.  Shave  a  large  area  of  the  abdominal  parietes. 

3.  Make  an  incision  through  the  skin  in  the  middle 
line  about  2  cm.  in  length,  midway  between  the  lower 
end  of  the  sternum  and  the  pubes. 

4.  Divide  the  aponeuroses  between  the  recti  upon  a 
director. 

5.  Divide  the  peritoneum  upon  a  director. 

6.  Introduce    the    inoculum    into    the    peritoneal 
cavity. 

7.  Close  the  peritoneal  cavity  with  Lembert's  su- 
tures. 

8.  Close  the  skin  and  aponeurosis  incisions  together 
with   interrupted  sutures  or  Michel's  steel  clips,  and 
apply  a  sealed  dressing. 

9.  Release  the  animal  from  the  operating  table. 

10.  Label,  etc. 

Suitable  sacs  may  be  readily  prepared  by  either  of 
the  following  methods' 

A.  Collodion  Sacs. 

i.  Dip  a  small  test-tube  (5  by  0.5  cm.),  bottom 
downward,  into  a  beaker  of  collodion,  and  dry  in  the 
air;  repeat  this  process  three  or  four  times. 


358  EXPERIMENTAL   INOCULATION    OF  ANIMALS 

2.  Dip  the  tube,  with  its  coating  of  collodion,  alter- 
nately into  a  beaker  of  alcohol  and  one  of  water.     This 
loosens  the  collodion  and  allows  it  to  be  peeled  off  in 
the  shape  of  a  small  test-tube. 

3.  Take  a  20  cm.  length  of  glass  tubing,  of  about 
the    diameter   of  the  test-tube  used  in  forming  the 
sac,  and   insert   one   end   into   the   open   mouth    of 
the  sac. 

4.  Suspend  the  glass  tube  with  attached  sac,  inside 
a  larger  test-tube,  by  packing  cotton- wool  in  the  mouth 
of  the  test-tube  around  the  glass  tubing,  and  place  in  the 
incubator  at   37°   C.    for  twenty-four   hours.     When 
removed  from  the  incubator,  the   sac  will  be   firmly 
adherent  to  the  extremity  of  the  glass  tubing. 

5.  Plug  the  open  end  of  the  glass  tubing  with  cotton- 
wool, and  sterilise  the  test-tube  and  its  contents  in  the 
steam  steriliser  oven. 

To  use  the  sac,  remove  the  plug  from  the  glass  tubing, 
partly  fill  the  sac  with  cultivation  to  be  inoculated,  by 
means  of  a  sterile  capillary  pipette,  and  replug  the 
tubing.  When  the  abdominal  cavity  has  been  opened, 
remove  the  tubing  and  attached  sac  from  the  protecting 
test-tube,  close  the  sac  by  tying  a  sterilised  silk  thread 
tightly  around  it  a  little  below  the  end  of  the  glass 
tubing,  and  separate  it  from  the  tubing  by  cutting 
through  the  collodion  above  the  ligature,  and  the  sac 
is  ready  for  insertion  in  the  peritoneal  cavity. 

B.  Celloidin  Sacs  (Harris). 
Materials  Required. 

Quill  glass  tubing. 

Gelatine  capsules  such  as  pharmacists  prepare  for  the  exhibition 
of  bulky  powders. 

Various  grades  of  celloidin,  thick  and  thin,  in  wide-mouthed 
bottles. 

1.  Take  a  piece  of  quill  glass  tubing  some  4  cm.  long 
by  5  mm.  diameter ;  heat  one  end  in  the  bunsen  flame. 

2.  Thrust  the  heated  end  of  the  tube  just  through 


CELLOIDIN    SACS 


359 


one  end  of  a  gelatine  capsule  and  allow  it  to  cool 
(Fig.  185). 

3 .  Remove  any  gelatine  from  the  lumen  of  the  tube 
with  a  heated  platinum  needle;  paint  the  joint  between 
capsule  and  tube  with  moderately  thick  celloidin  and 
allow  to  dry. 

4.  Dip  the  capsule  into  a  beaker  containing  thin 
celloidin,  beyond  the  junction  with  the  glass  and  after 
removal  rotate  it  in  front  of  the  blow- 
pipe air  blast  to  dry  it  evenly.     Repeat 

these  manoeuvres  until  a  sufficiently  thick 
coating  is  obtained. 

5.  Apply  thick  celloidin  to  the  tube- 
capsule  joint,  the  opposite  end  of  the  cap- 
sule, and  the  line  of  junction  of  the  cap- 
sule with  its  cap ;  dry  thoroughly. 

6.  With  a  teat  pipette  fill  the  capsule 
(through  the  attached  tube)    with  hot 
water,  and  stand  the  capsule  in  a  beaker 
of   boiling  water  for  a  few  minutes  to 
melt  the  gelatine. 

7 .  Remove  the  solution  of  gelatine  from 

the  interior  of  the  celloidin  case  with  a  pipette. 

8.  Fill  the  sac  with  nutrient  broth  and  place  it, 
glass  tube  downward,  in  a  tube  containing  sufficient 
sterile  nutrient  broth  to  cover  the  sac  to  the  depth 
of  i  cm.     Plug  the  tube  and  sterilise  in  the  steamer 
in  the  usual  manner. 

9.  To  prepare  the  sac  for  use,  empty  it  out  of  the 
broth  tube  into  a  sterile  glass  dish. 

10.  Grasp  the  tube  near  its  junction  with  the  sac 
in  the  jaws  of  sterile  forceps,  and  with  a  teat  pipette 
remove  sufficient  of  the  contained  broth  to  leave  a 
small  space  in  the  sac.     Introduce  the  inoculum  in  the 
form  of  an  emulsion  by  means  of  another  pipette. 

11.  Still  holding  the  tube  in  the  forceps,  draw  it 
out  and  seal  off  near  the  sac  in  the  blowpipe  flame. 


Fig. 
'   un 

capsules. 


185.— 
Making  celloidin 


360  EXPERIMENTAL   INOCULATION    OF  ANIMALS 

12.  When  cool  wash  the  sac  in  sterile  water,  then 
transfer  to  a  tube  of  nutrient   broth  and   incubate 
over  night  to  determine  its  impermeability  to  bacteria. 

13.  If  the   broth  outside  the  sac  remains   sterile, 
insert  the  sac  in  the  peritoneal  cavity  of  the  experi- 
mental animal. 

5.  Intracranial. — (Anesthetic,  A.  C.  E.) 
Trephines  and  Surgical  Engine. — The  most  useful 
instrument  for  intracranial  operations  upon  animals 
is  the  small  nasal  trephine  (Curtis)  having  a  tooth  cut- 


FIG.  1 86. — Guarded  trephine. 

ting  circle  of  7  mm.  The  addition  of  an  adjustable 
collar  guard — secured  by  a  screw — prevents  acciden- 
tal laceration  of  the  dura  mater  or  brain  substance1 
(Fig.  1 86) .  This  size  is  suitable  for  monkeys,  dogs,  cats 
and  large  rabbits.  Other  smaller  sizes  which  will  be 
found  useful  for  guinea  pigs  and  other  small  animals 
cut  circles  of  6  and  4  mm. ;  for  very  small  animals — 
young  guinea  pigs  and  rats — a  small  dental  drill  or 
screw  will  make  a  sufficiently  large  hole  to  admit  the 
syringe  needle.  The  trephine  can  be  set  in  ordinary 
metal  handles  and  rotated  by  hand,  but  a  surgical 
engine  of  some  kind  is  much  preferable  on  the  score 
of  rapidity  and  safety  to  the  animal.  The  Guy's  elec- 
trical Dental  engine2  (Fig.  187)  which  can  be  connected 
to  a  lamp  socket  or  wall  plug,  and  is  operated  by  a  foot 
switch,  although  inexpensive  is  eminently  satisfactory. 

NOTE. — A  fine  dental  drill  attached  to  the  dental  engine  renders 
the  manufacture  of  aluminium  handles  needles  (see  page  71) 
quite  an  easy  matter. 

1  This  modification  is  made  for  the  author  by  Messrs.  Down  Bros.,  St. 
Thomas's  Street,  London,  S.  E. 

2  Manufactured   by   Messrs.  Francis  Lepper,    56,    Great   Marlborough 
Street,  London,  W. 


INTRACRANIAL  361 

(a)  Subdural. 

1.  Anaesthetise  the  animal  and  secure  it  to  the  oper- 
ating table,  dorsum  uppermost. 

2.  Shave  a  portion  of  the  scalp  immediately  in  front 
of  the  ears. 

3.  Mark  out  with  a  sharp  scalpel  a  crescentic  flap 


FlG.  187. — Guy's  electrical  dental  engine. 

of  skin  muscle,  etc.,  convexity  forward,  commencing 
0.5  cm.  in  front  of  the  root  of  one  ear  and  terminating 
at  a  similar  spot  in  front  of  the  other  ear.  Reflect  the 
marked  flap. 

4.  Make  a  corresponding  incision  through  the  perios- 
teum and  raise  it  with  a  blunt  dissector. 

5.  With  a  small  trephine  (diameter  6  mm.)  remove 
a  circular  piece  of  bone  from  the  parietal  segment. 
The  centre  of  the  trephine  hole  should  be  at  the  inter- 
section of  the  median  line  and  a  line  joining  the  pos- 
terior canthi  (Fig.  188). 

6.  Introduce  the  inoculum   by  means  of  a  hypo- 
dermic syringe,  perforating  the  dura  mater  with  the 
needle  and  depositing  the  material  immediately  below 
this  membrane,  at  the  same  time  taking  care  to  avoid 
injuring  the  sinuses. 


362 


EXPERIMENTAL   INOCULATION    OF  ANIMALS 


7.  Turn  back  the  flap  of  skin  and  secure  it  in  position 
with  Michel's  steel  clips. 

8.  Dress  with  sterile  gauze  and  wool  and  seal  the 
dressing  with  collodion. 

9.  Label,  etc. 

(b)  Intracerebral. — This  inoculation  is  performed 
precisely  as  for  subdural  save  in  step 
6  the  needle  after  perforating  the  dura 
mater  is  pushed  onward  into  the  sub- 
stance of  one  or  other  cerebral  hemis- 
pheres before  the  contents  are  ejected. 

6.  Intraocular. — 

(a)    Fluid    Inoculum. — (Anesthetic, 
cocaine.) 

1 .  Instil  a  few  drops  of  a  sterile  solu- 
tion of  cocaine,  and  repeat  the  instilla- 
tion in  two  minutes. 

2 .  Five  minutes  later  have  the  animal 
firmly  held  by  an  assistant  as  in  intra- 
venous injection  (see  Fig.  189)  the  head 
being  steadied  by  the  assistant's  hands. 

3 .  Select  two  needles  to  accurately  fit 
the  same  syringe  and  sterilise. 

trephine        4.  Attach  one  needle  to  the  syringe 
and  take  up  the  required  dose  of  inocu- 
lum and  remove  the  needle. 

5.  Steady  the  eye  with  fixation  forceps;  then  pierce 
the  cornea  with  the  other  syringe  needle  and  allow  the 
aqueous  to  escape  through  the  needle. 

6.  Without  removing  the  needle  from  the  cornea 
attach  the  syringe  and  make  the   injection  into  the 
anterior  chamber. 

7.  Irrigate  the  conjunctival  sac  with  sterile  saline 
solution. 

8.  Label,  etc. 

(b)  Solid  Inoculum. — (Anesthetic,  A.  C.  E.) 


FIG.  1 88.— In- 
tracranial  inocula- 
t  i  o  n  of  rabbit. 


of     the 
hole. 


INTRAVENOUS  363 

1.  Anaesthetise  the  animal  and  secure  it  firmly  to 
the  operating  table. 

2.  Irrigate   the    conjunctival   sac   thoroughly   with 
sterile  saline  solution. 

3.  Make  an  incision  through  the  upper  quadrant  of 
the  cornea  into  the  anterior  chamber  by  means  of  a 
triangular  keratome. 

4.  Separate  the  lips  of  the  corneal  wound  with  a 
flexible  silver  spatula;  seize  the  solid  inoculum  in  a 
pair  of  iris  forceps,  introduce  it  through  the  corneal 
wound,  and  deposit  it  on  the  anterior  surface  of  the 
iris;  withdraw  the  forceps. 

5.  Again  irrigate  the  sac  and  the  surface  of  the  cor- 
nea. 

6.  Release  the  animal  from  the  operating  table. 

7.  Label,  etc. 

7.  Intrapulmonary. — 

Fluid  Inoculum. — (Anesthetic,  none.) 

1.  Have  the  animal  firmly  held  by  an  assistant. 
(In  this  case  the  foreleg  of  the  selected  side  is  drawn  up 
by  the  assistant  and  held  with  the  ear  of  that  side.) 

2.  Shave  carefully  in  the  axillary  line  and  disinfect 
the  denuded  skin. 

3.  Thrust  the  needle  of  the  syringe  boldly  through 
the  fifth  or  sixth  intercostal  space  into  the  lung  tissue. 

4.  Inject  the  contents  of  the  syringe  slowly. 

5.  Label,  etc. 

8.  Intravenous. — 

Fluid  Inoculum. — (Anesthetic,  none.) 

The  site  selected  for  the  injection  in  the  rabbit  is  the 
posterior  auricular  vein  (see  Fig.  192).  Although  this 
is  smaller  than  the  median  vein,  it  is  firmly  bound  down 
to  the  cartilage  of  the  ear  by  dense  connective  tissue, 
and  is  therefore  more  readily  accessible .  (In  the  guinea- 
pig  the  jugular  vein  must  be  utilised,  and  in  order  to 
perform  the  inoculation  satisfactorily  a  general  anaes- 


364  EXPERIMENTAL   INOCULATION    OF  ANIMALS 

thetic  must  be  administered  to  the  animal.  In  the 
monkey  or  the  dog,  the  internal  saphenous  vein  is  the 
most  convenient  and  before  puncturing  should  be  dis- 
tended or  rendered  prominent  by  compressing  the  vein 
above  the  selected  site.) 

Preparation  of  the  Inoculum. — Care  must  be  taken 
in  preparing  the  inoculum,  as  the  injection  of  even 
small  fragments  may  cause  fatal  embolism.  To  obviate 
this  risk  the  fluid  should,  if  possible,  be  filtered  through 
sterile  filter  paper  before  filling  into  the  syringe. 

Air  bubbles,  when  injected  into  a  vein,  frequently 
cause  immediate  death.  To  prevent  this,  the  syringe 
after  being  filled  should  be  held  in  the  vertical  posi- 
tion, needle  uppermost.  A  piece  of  sterile  filter  paper 
is  then  impaled  on  the  needle  and  the  piston  of  the 
syringe  pressed  upward  until  all  the  air  is  expelled  from 
the  barrel  and  needle.  Should  any  drops  of  the  inocu- 
lum be  forced  out,  they  will  fall  on  the  filter  paper, 
which  should  be  immediately  burned. 

1.  Have  the  animal  firmly  held  by  an  assistant. 
The  selected  ear  is  grasped  at  its  root  and  stretched 
forward  toward  the  operator. 

2.  Shave  the  posterior  border  of  the  dorsum  of  the 
ear. 

3.  Disinfect   the    skin   over   the    vein,    rubbing   it 
vigourously  with  cotton- wool  soaked   in    lysol.     The 
friction  will  make  the  vein  more  conspicuous.     Wash 
the  lysol  off  with  ether  and  allow  the  latter  to  evaporate. 

4.  Direct  the  assistant  to  compress  the  vein  at  the 
root  of  the  ear.     This  will  cause  its  peripheral  portion 
to  swell  up  and  increase  in  calibre. 

5.  Hold  the  syringe  as  one  would  a  pen  and  thrust 
the  point  of  the  needle  through  the  skin  and  the  wall 
of  the  vein  till  it  enters  the  lumen  of  the  vein  (Fig.  189) . 
Now  press  it  onward  in  the   direction  of  the   blood 
stream — i.  e.,  toward  the  body  of  the  animal. 

6.  Direct   the   assistant   to   cease   compressing   the 


INHALATION  365 

root  of  the  ear,  and  slowly  inject  the  inoculum.  (If 
the  fluid  is  being  forced  into  the  subcutaneous  tissue, 
a  condition  which  is  at  once  indicated  by  the  swelling 
that  occurs,  the  injection  must  be  stopped  and  another 
attempt  made  at  a  spot  closer  to  the  root  of  the  ear  or 
at  some  point  on  the  corresponding  vein  on  the  opposite 
ear.) 

7 .  Withdraw  the  needle  and  press  a  pledget  of  cotton- 
wool over  the  puncture  to  ensure  closure  of  the  aperture 
in  the  vein  wall. 

8.  Label,  etc. 


FIG.  189. — Intravenous  inoculation. 

9.  Inhalation. — 

(a)  Fluid  Inoculum. — (Anesthetic,  none.) 

1.  Place  the  animal  in  a  closed  metal  box. 

2.  Through  a  hole  in  one  side  introduce  the  nozzle 
of  some  simple  spraying  apparatus,  such  as  is  used  for 
nasal  medicaments. 

3.  Fill  the  reservoir  of  the  instrument   (previously 
sterilised)    with  the  fluid  inoculum,   and  having  at- 
tached the  bellows,  spray  the  inoculum  into  the  interior 
of  the  box. 

4.  On  the  completion  of  the  spraying,  open  the  box, 
spray  the  animal  thoroughly  with  a  10  per  cent,  solution 
of  formaldehyde  (to  destroy  any  of  the  virus  that  may 
be  adhering  to  fur  or  feathers). 

5.  Transfer  the  animal  to  its  cage. 


366  EXPERIMENTAL   INOCULATION    OF  ANIMALS 

6.  Label,  etc. 

7.  Thoroughly  disinfect  the  inhalation  chamber. 

(b)  Fluid  or  Powdered  Inoculum. — A  n&sthetic,  A.C.E. 
i.  Anaesthetise  the  animal  and  secure  it  firmly  to 
the  operating  table. 


FIG.  190. — Gag  for  rabbits. 

2.  Prop  open  the  mouth  by  means  of  some  form  of 
gag ;  seize  the  tongue  with  a  pair  of  forceps  and  draw  it 
forward. 

The  most  convenient  form  of  gag  for  the  rabbit  or 
cat  is  that  shown  in  Fig.  190.  It  is  simply  a  strip  of 
hard  wood  shaped  at  the  middle  and  provided  with  a 
square  orifice  through  which  a  tracheal  or  cesophageal 
tube  can  be  passed. 

3.  Pass  a  previously  sterilised   glass   tube    (17  cm. 
long,  0.5  cm.  diameter,  with  its  terminal  2  cm.  slightly 
curved)  down  through  the  larynx  into  the  trachea. 

4.  Connect  the  straight  portion  of  a  Y-shaped  piece 
of  tubing  to  the  upper  end  of  the  sterilised  tube  and 
couple  one  branch  of  the  Y  to  a  separatory  funnel  con- 
taining the  fluid  inoculum,  or  insufflator  containing  the 
powdered  inoculum,  and  the  other  to  a  hand  bellows. 

5.  Allow  the  fluid  inoculum  to  run  into  the  lungs  by 
gravity,  or  blow  in  the  powdered  inoculum  by  means 
of  a  rubber-ball  bellows. 

6.  Remove  the  intratracheal  tube ;  release  the  animal 
from  the  table. 

7.  Label,  etc. 

As  an  alternative  method  in  the  case  of  fairly  large 
animals,  such  as  rabbits,  etc.,  a  sterile  piece  of  glass 
tubing  of  suitable  diameter  may  be  passed  through 
the  larynx  down  the  trachea  almost  to  its  bifurcation. 


INTRAGASTRIC  367 

Fluid  cultivations  may  then  be  literally  poured  into 
the  lungs,  or  cultivations,  dried  and  powdered,  may 
be  blown  into  the  lung  by  the  aid  of  a  small  hand 
bellows  or  even  a  teat  pipette. 

10.  Intragastric  Inoculation. — Fluid  or  semifluid  in- 
oculum. (Anaesthetic  none.) 

The  method  of  performing  the  operation  is  varied 
slightly  according  to  the  size  of  the  experimental  animal. 

A.  Monkey,  Rabbit,  Guinea-pig. 

1.  Secure  the  animal  to  the  operating  table  ventral 
surface  uppermost. 

2 .  Prop  the  mouth  open  with  a  gag ;  draw  the  tongue 
forward  with  forceps. 

3.  Sterilise  a  soft  rubber  catheter  (No.  10  or  8  Eng- 
lish scale,  or  No.  18  or  15  French)  and  lubricate  it  with 
sterile  glycerine. 

4.  Pass  it  to  the  back  of  the  pharynx,  keeping  the 
end  in  the  middle  line. 

5.  Gently  assist  the  progress  of  the  catheter  down  the 
oesophagus  until  it  passes  the  cardiac  orifice  of  the 
stomach.     Do  not  use  any  force. 

6.  Take  up  the  required  dose  of  inoculum  into  a 
sterilised   pipette.     Insert   the   point   of   the   pipette 
into  the  open  end  of  the  catheter  and  allow  the  fluid 
to  run  down  into  the  stomach.     Remove  the  pipette 
and  drop  it  into  a  jar  of  lysol. 

7.  With  another  sterile  pipette  run  one  cubic  cen- 
timetre of  sterile  saline  solution  through  the  catheter 
to  wash  out  the  last  traces  of  the  inoculum. 

8.  Withdraw  the  catheter. 

9.  Label,  etc. 

B.  Rats  and  Mice  (Mark's  Method). 

i.  Secure  the  animal  in  the  vertical  position. 

(a)  Rat. — Take  a  pair  of  catch  sinus  forceps  about 
22  cm.  in  length  and  seize  the  animal  by  the  loose  skin 
of  the  head  as  far  forward  as  possible — fix  the  forceps, 
and  holding  the  instrument  vertically  upward,  transfer 


368  EXPERIMENTAL   INOCULATION    OF  ANIMALS 

to  the  left  hand  of  an  assistant  who  secures  the  animal's 
tail  between  the  fingers  grasping  the  handle  of  the  for- 
ceps. (See  Fig.  191.) 

(b)  Mouse. — An  assistant  grasps  the  loose  skin 
between  the  ears  as  far  forwards  as  possible  between 
the  fore-finger  and  thumb  of  the  left  hand.  He  now 


FIG.  191. — Intragastric  inoculation  of  rat. 

grasps  the  tail  with  the  right  hand,  draws  the  mouse 
straight  and  passes  the  tail  between  the  fourth  and  little 
ringers  of  the  left  hand  and  secures  it  there. 

2.  The  assistant  takes  a  closed  pair  of  thin-b laded 
forceps  in  his  right  hand,  passes  the  ends  into  the 
animal's  mouth,  then  allows  the  blades  to  separate. 
This  opens  the  animal's  jaw  and  serves  as  a  gag. 


FEEDING  369 

3 .  Moisten  the  sterilised  oesophageal  tube  with  sterile 
water.     (This  tube  is  of  silk  rubber,  6.5  cm.  in  length, 
with  the  distal  end  rounded,  the  proximal  end  mounted 
in  a  syringe  needle  head,  which  fits  the  nozzles  of  the 
two  sterile  syringes  to  be  used.) 

4.  Grasp  the  tube  about  its  middle  and  pass  it  into 
the  animal's  mouth,  downwards  and  a  little  to  one  side 
or  the  other  until  its  length  is  lost  in  the  digestive  tract 
and  mouth.     Gentle  guidance  is  alone  necessary.     Do 
not  use  any  force. 

5.  Take  up  the  required  dose  of  inoculum  into  the 
syringe ;  insert  the  nozzle  of  the  syringe  into  the  needle- 
mount,  and  force  the  piston  down. 

6.  Steadying  the  needle-mount  with  the  left  hand, 
detach  the  syringe. 

7.  Draw  up  some  sterile  water  in  the  second  (sterile) 
syringe,  and  inserting  its  nozzle  into  the  needle-mount 
force  a  few  drops  of  water  through  the  tube  to  wash  it 
out. 

8.  With  one  quick  upward  movement  remove  the 
tube  from  the  animal's  mouth. 

9.  Label,  etc. 

One  other  method  of  inoculation  remains  to  be 
described,  which  does  not  require  operative  inter- 
ference. 

11.  Feeding.— 

1.  Fluid  Inoculum. — Small  pieces  of  sterilised  bread 
or  sop  (sterilised  in  the  steamer  at  100°  C.)  are  soaked 
in  the  fluid  inoculum  and  offered  to  the  animals  in  a 
sterile  Petri  dish  or  capsule. 

2.  Solid  Inoculum. — Small  pieces  of  tissue  are  placed 
in  sterile  vessels  and  offered  to  the  animals. 


XVIII.     THE  STUDY  OF  EXPERIMENTAL 
INFECTIONS  DURING  LIFE. 

The  possession  of  pathogenetic  properties  by  an 
organism  under  study  is  indicated  by  the  "infection" 
of  the  experimental  animal — a  term  which  is  employed 
to  summarise  the  condition  resulting  from  the  success- 
ful invasion  of  the  tissues  of  the  experimental  animal 
by  the  micro-organisms  inoculated  and  by  their  multi- 
plication therein.  Infection  is  considered  to  have 
taken  place : 

1.  When  the  death  of  the  animal  is  produced  as  a 
direct  consequence  of  the  inoculation. 

2.  When  without  necessarily  producing  death  the 
inoculation  causes  local  or  general  changes  of  a  patho- 
logical character. 

3.  When  either  with  or  without  death,  or  local  or 
general   changes   occurring,    certain   substances  make 
their  appearance  in  the  body  fluids,    which  can   be 
shown  (in  vitro  or  in  vivo)  to  exert  some  profound  and 
specific  effect  when  brought  into  contact  with  sub- 
cultivations  of  the  organism  originally  inoculated. 

The  important  factors  in  the  production  of  infection 
are: 

A.  Seed.     Virulence  of  organism. 

Dose  of  organism. 

B.  Soil.      Resistance   offered    by  the  cells    of   the 

experimental  animal. 

The  first  two  factors,  although  variable,  are  to  a 
certain  extent  under  the  control  of  the  experimenter. 
Thus  by  suitable  means  the  virulence  of  an  organism 


GENERAL    OBSERVATIONS  371 

can  be  exalted  or  attenuated,  whilst  the  size  of  the  dose 
may  be  increased  or  diminished.  The  third  factor 
also  varies,  not  only  amongst  different  species  of 
animals,  but  also  amongst  different  individuals  of  the 
same  species.  The  essential  causes  of  this  variation 
are  not  so  obvious,  so  that  beyond  selecting  the  animals 
intended  for  similar  experiments  with  regard  to  such 
points  as  age,  size  or  sex,  but  little  can  be  done  to 
standardise  cell  resistance. 

Immediately  an  animal  has  been  inoculated  a  period 
of  clinical  observation  must  be  entered  upon,  which 
should  only  terminate  with  the  death  of  the  animal. 
The  general  observations  should  at  first  and  if  the 
infection  is  an  acute  one,  be  made  daily — later,  and  if 
the  animal  appears  to  be  unaffected  or  if  the  infection 
is  chronic,  both  general  and  special  observations 
should  be  carried  out  at  weekly  intervals.  If  the  ani- 
mal appears  to  be  still  unaffected,  it  should  be  killed 
with  chloroform  vapour  at  the  end  of  two  or  three 
months  and  a  complete  post-mortem  carried  out. 

A.  The  general  observations  should  take  cognisance 
of: 

1.  General    appearance.     The    experimental    animal 
should  be  inspected  daily,  not  only  with  a  view  to 
detecting  symptoms  due  to  the  experimental  infection, 
but  also  to  prevent  any  intercurrent  infection,  natur- 
ally acquired,  from  escaping  notice  (wde  page  337). 

2.  The  weight  of  the  inoculated  animal  should  be 
observed  and  recorded  each  day  during  the  course 
of  an  experimental  infection  at  precisely  the  same  hour, 
preferably  just  before  the  morning  feed. 

3.  The    temperature    should    similarly    be    recorded 
daily,  if  not  more  frequently,  during  the  whole  period 
the  animal  is  under  observation,  and  carefully  charted 
— individual  variations  will  at  once  become  apparent. 
It  should  be  borne  in  mind  that  the  temperature  re- 
garded as  normal  for  man  (37.5°  C.)  is  not  the  normal 


372 


EXPERIMENTAL   INFECTIONS    DURING    LIFE 


average  temperature  of  any  of  the  lower  animals  save 
the  rat  and  mouse.  The  accompanying  table  of  nor- 
mal averages  for  the  animals  usually  employed  in  bac- 
teriological research  may  be  of  use  in  preventing  the 
erroneous  assumption  that  pyrexia  is  present  in  an  ani- 
mal, which  merely  shows  its  own  normal  temperature. 

NORMAL  AVERAGES. 


Rectal 

Pulse. 

Respirations. 

Animal. 

Temp.  °C. 

Rate  pe 

r  minute. 

Frog.  . 

8.  Q—  17.  2 

80 

12 

Mouse         

•27.4 

I2O 

Rat 

37  .  e 

2  IO 

Guinea  pig  

38.6 

i^o 

80 

Rabbit   

38.7 

I^C 

ec 

Cat 

38    7 

I^O 

24 

Dog. 

38.6 

CK 

1C 

Goat             .... 

40  .  o 

7C 

16 

Ox 

38.8 

A? 

Horse     

37  .  Q 

38 

II 

Monkey  (Rhesus)  .  . 
Pigeon 

38.4 
40  o 

100 

1*6 

19 

30 

Fowl   

41.6 

140 

12 

B.  Special  observations  comprise  some  or  all  of  the 
following,  according  to  the  method  of  inoculation  and 
the  character  of  the  virus. 

1.  The  site  of  inoculation  should  be  minutely  exam- 
ined at  least  at  weekly  intervals,  and  the  neighbouring 
lymphatic  glands  palpated. 

2.  Any  local  reaction  at  the  site  of  inoculation  and 
any  other  readily  accessible  lesion  should  be  carefully 
investigated.     Any   suppurative   process   which   may 
occur,    whether   in    the    subcutaneous    tissues    or    in 
joints,  should  be  explored  and  the  pus  carefully  ex- 
amined both  microscopically  and  culturally. 


SPECIAL    OBSERVATIONS  373 

Fluid  secretions  and  excretions,  such  as  pus  or 
serous  exudates  when  accessible  are  collected  direct 
from  the  body  in  sterile  capillary  pipettes  (vide  Fig. 
I3&,)  in  the  following  manner: 

i.  Open  the  case  containing  the  pipettes,  grasp  one 
by  the  plugged  end,  remove  it  from  the  case,  and 
replace  the  lid  of  the  latter. 

2. Attach  a  rubber  teat  (vide  page  10)  to  the  plugged 
end  of  the  pipette  and  use  the  teat  as  the  handle  of  the 
pipette. 

3.  Pass  the  entire  length  of  the  pipette  twice  or 
thrice  through  the  flame  of  the  Bunsen  burner. 

4.  Snap  off  the  sealed  end  of  the  pipette  with  a  pair 
of  sterile  forceps. 

5.  Compress  the  india-rubber  teat,  thrust  the  point 
of  the  pipette  into  the  secretion ;  now  relax  the  pressure 
on  the  teat  and  allow  the  pipette  to  fill. 

6.  Remove  the  point  of  the  pipette  from  the  secretion, 
allow  the  fluid  to  run  a  short  distance  up  the  capillary 
stem  and  seal  the  point  of  the  pipette  in  the  flame.     (If 
using  a  pipette  with  a  constriction  below  the  plugged 
mouthpiece  (Fig  136),  this  portion  of  the  pipette  may 
also  be  sealed  in  the  flame.) 

When  ready  to  examine  the  morbid  material  snap 
off  the  sealed  end  of  the  pipette  with  sterile  forceps  and 
eject  the  contents  of  the  pipette  into  a  sterile  capsule. 
The  material  can  now  be  utilized  for  coverslip  prep- 
arations, cultivations  and  inoculation  experiment. 

3.  The  peripheral  blood  should  be  examined  from 
time  to  time  for  from  this  tissue  is  often  obtained 
the  fullest  information  as  to  the  course  and  progress 
of  an  infection. 

a.  The  histological  examination  of  the  blood  should 
be  directed  chiefly  to  observations  on  the  number  and 
kind  of  white  cells;  and  since  but  few  bacteriologists 
are  at  the  same  time  expert  comparative  haematolo- 
gists,  some  notes  on  the  normal  characters  of  the  blood 


374 


EXPERIMENTAL   INFECTIONS    DURING    LIFE 


of  the  commoner  laboratory  animals,  contrasted  with 
those  of  man,  are  inserted  for  reference.  These  have 
been  very  kindly  compiled  for  me  by  my  friend  and  one 
time  colleague  Dr.  Cecil  Price  Jones. 

COMPARATIVE    H^EMOCYTOLOGY   OF    LABORATORY 
ANIMALS. 


Totals 

Percentages 

Animal 

Lympho- 

Large 

Poly- 

Eosin- 

Mast 

Red  cells 

White 

Hb,  pec- 

cytes, 

monos, 

morph, 

oph, 

cells, 

cells 

cent. 

per 

per 

per 

per 

per 

cent. 

cent. 

cent. 

cent. 

cent. 

Frog  

490,000 

8,000 

58 

40 

10.  O 

22  .0 

IS 

13 

Mouse.  .  . 

8,700,000 

8,000 

78 

60 

21.5 

17.0 

i  .4 

o.  r 

Rat 

9,000,000 

9,000 

85 

54 

7  .  o 

37-5 

I  t  O 

O  .  2 

Guinea- 

pig  

5,700,000 

10,000 

99 

55 

9-0 

32.8 

3-0 

O.  2 

Rabbit.... 

6,000,000 

7,000 

70 

50 

2.0 

46.0 

0.6 

1.4 

Rhesus... 

4,500,000 

13,000 

77 

43 

5-o 

50.0 

1.3 

o.  7 

Goat  

14,600,000 

15,000 

58 

35 

6.3 

56.7 

1.25 

o.7S 

Fowl  

3,500,000 

30,000 

100 

49 

3-0 

42.0 

I  .0 

5-0 

Pigeon.  .  . 

3,500,000 

20,000 

101 

43 

9.0 

43-0 

3-0 

2  .0 

Man 

(adult).. 

5,000,000 

7,500 

100 

25 

5-5 

65 

4.0 

0.5 

Normal 

(4.5-5) 

(7-9) 

(95-101) 

(20-30) 

(4-8) 

(55-68) 

(3-5) 

(0.5-2) 

limits. 

millions. 

thou- 

sands. 

The  above  table  represents  in  each  case  the  average 
of  a  large  number  of  counts. 

REMARKS. 

Frog. — The  red  cells  are  large  oval  nucleated  (20-25/1 
by  12-15^  discs,  the  nucleus  relatively  small  and 
irregularly  elongated  or  oval,  about  io/*  in  length. 
Many  primitive  and  developing  forms  are  usually  ob- 
served— also  free  nuclei  and  many  cells  in  various  stages 
of  degeneration.  Haemoglobin  estimation  is  difficult 
owing  to  turbidity  of  the  blood  after  dilution  with 
water.  The  polymorphonuclear  leucocytes  are  large 


COMPARATIVE   H^MATOLOGY  375 

cells,  about  20^;  no  definite  granules  can  be  observed. 
The  eosinophile  cells  contain  large  deeply  staining 
coccal-shaped  granules. 

Mouse. — The  granules  of  the  polymorphonuclear 
leucocytes  are  usually  not  stained,  or  only  very  faintly, 
so.  The  nucleus  of  the  eosinophile  cell  is  ring-shaped 
or  much  divided,  and  the  granules  are  coccal  and  stain 
oxyphile.  The  remarkable  character  of  the  blood  is 
the  high  percentage  of  large  mononudear  cells. 

Rat. — The  fine  rod-shaped  granules  of  the  poly- 
morphonuclear leucocytes  are  usually  very  faintly 
stained.  The  granules  of  eosinophile  cells  are  well 
stained  and  coccal-shaped,  the  nucleus  is  often  ring 
shaped.  The  basophile  granular  cells  are  few — but  the 
granules  are  large,  and  stain  deeply  basophile. 

Guinea-pig. — Polychromasia  and  punctate  baso- 
philia  of  red  cells  are  very  commonly  observed — 
nucleated  red  cells  are  also  frequent.  The  large  mono- 
nuclear  cells  often  contain  vacuoles — "  Kurlow  cells  " — 
possibly  of  a  parasitic  nature. 

Rabbit. — It  is  not  uncommon  to  find  nucleated 
red  cells  in  films  from  quite  healthy  animals.  The 
granules  of  the  polymorphonuclear  leucocytes  stain 
oxyphile.  The  coarse  granules  of  the  eosinophile  cells 
appear  to  stain  less  deeply  oxyphile,  probably  owing  to 
the  basophile  staining  of  the  cytoplasm. 

Rhesus  monkey. — The  blood  cells  resemble  those 
met  with  in  human  blood.  The  minute  neutrophile 
granules  of  the  polymorphonuclear  leucocytes  are  often 
very  scanty,  and  sometimes  apparently  absent.  The 
eosinophile  cells  are  not  so  densely  packed  with  coarse 
oxpyhile  granules  as  in  the  human  eosinophile,  and  the 
nuclei  of  these  cells  are  usually  much  divided,  or 
polymorphous . 

Goat. — The  red  cells  are  small,  nonnucleated  discs, 
only  about  4.5^  diameter,  not  much  more  than  half 
that  of  the  human  red  cell.  The  polymorphonuclear 


376  EXPERIMENTAL   INFECTIONS   DURING   LIFE 

leucocytes  have  only  a  few  very  minute  coccal-shaped 
oxyphile  granules,  the  nucleus  is  polymorphous.  The 
eosinophile  cells  are  large  cells  up  to  20^,  the  cyto- 
plasm is  basophile  and  contains  coarse  coccal-shaped 
oxyphile  granules,  and  the  nucleus  is  often  much 
divided. 

Fowl. — The  red  cells  are  oval  nucleated  discs  about 
I2/*  by  6fi,  the  nucleus  being  relatively  small  (about 
4/£  long),  irregularly  elongated  or  oval;  round,  more 
deeply  stained  cells  with  round  or  diffuse  nuclei,  also 
free  nuclei  and  degenerated  forms  of  red  cells  are  often 
present.  The  granules  of  the  cells  corresponding  to 
the  polymorphonudear  leucocytes  are  rod-shaped,  often 
beaded  or  with  clubbed  ends.  The  nucleus  is  not 
polymorphous,  but  usually  divided  into  two,  though 
it  may  be  single.  The  cells  probably  corresponding  to 
eosinophile  leucocytes  have  fine  coccal-shaped  granules, 
faintly  staining  eosinophile  or  neutrophile.  The  baso- 
phile granules  of  the  "mast"  cells  are  coccal-shaped,  of 
various  size — often  quite  powdery. 

Pigeon. — Red  cells  resemble  those  of  the  fowl,  and 
similar  varieties  of  appearance  may  be  noted.  The 
granules  of  those  cells  which  correspond  to  polymorpho- 
nuclear  leucocytes  are  rod-shaped,  but  smaller  and 
finer  than  in  the  fowl,  and  do  not  show  clubbed  ap- 
pearances. The  nucleus  is  not  polymorphous,  and  only 
occasionally  divided.  The  coccal-shaped  granules  of 
the  eosinophile  cells  are  stained  more  deeply  oxyphile 
than  those  of  the  corresponding  cells  of  the  fowl. 

The  preparation  of  dried  films  for  this  histological 
examination  of  the  blood  is  carried  out  as  follows : 

i.  Small  samples  of  blood  for  the  preparation  of 
blood  films  are  most  conveniently  obtained  from  the 
veins  of  the  ear  in  most  of  the  ordinary  laboratory 
animals,  viz.,  monkey,  goat,  dog,  cat,  rabbit,  guinea- 
pig  ;  in  the  pigeon  and  fowl  the  axillary  vein  should  be 
punctured;  in  the  rat  and  mouse  either  a  vein  in  the 


EXAMINATION    OF    BLOOD  377 

ear  or  preferably  by  wounding  the  tip  of  the  tail ;  in  the 
frog,  the  web  of  the  foot  should  be  selected. 

2.  Puncture  the  selected  vein  with  a  sharp  needle. 
A  flat  Hagedorn  needle  (size  No.  8)  with   a   cutting 
edge  is  the  most  useful  for  this  purpose.     If  the  vein 
cannot  be  distended  by  proximal  compression,  vigourous 
friction  with  a  piece  of  dry  lint  may  have  the  desired 
effect — or  a  test-tube  full  of  water  at  about  40°  C. 
may  be  placed  close  to  the  vein.     Failing  these  methods, 
a  drop  or  two  of  xylol  may  be  dropped  on  the  skin  just 
over  the  vein,  left  on  for  a  few  seconds  and  then  wiped 
off  with  a  piece  of  dry  lint. 

3.  One  of  the  short  ends  of  a  3  by  i  glass  slip  is 
brought  into  contact  with  the  exuding  drop  of  blood, 
so  that  it  picks  up  a  small  drop. 

4.  The  slide  is  then  lowered  transversely  on  to  the 
surface  of  a  second  3  by  i  slip,  which  rests  on  the 
bench  near  to  one  end  at  an  angle  of  about  45°,  and 
retained  in  this  position  for  a  few  seconds,  while  the 
drop  of  blood  spreads  along  the  whole  of  the  line  of 
contact  (see  also  Fig.  69). 

5.  Draw  the  first  slide  firmly  and  evenly  along  the 
entire  length  of  the  lower  slide,  leaving  a  thin  regular 
film  which  will  probably  show  the  blood  cells  only  one 
layer  thick. 

6.  Allow  the  film  to  dry  in  the  air. 

7.  Stain  with  one  of  the  polychrome  blood  stains 
(seepage  97). 

8.  Examine  microscopically. 

b.  The  bacteriological  examination  of  the  blood  is 
directed  solely  to  the  demonstration  of  the  presence  in 
the  circulating  blood  of  the  organisms  previously  in- 
jected into  the  animal.  For  this  purpose  several  cubic 
centimetres  of  blood  should  be  taken  in  an  all-glass 
syringe  from  an  accessible  vein  corresponding  to  one  of 
those  suggested  as  the  site  of  intravenous  inoculation — 
and  under  similar  aseptic  precautions. 


378  EXPERIMENTAL   INFECTIONS   DURING   LIFE 

1.  Sterilise  an  all-glass  syringe  of  suitable  size,  and 
when  cool  draw  into  the  syringe  some  sterile  sodium 
citrate  solution  and  moisten  the  whole  of  the  interior  of 
the  barrel ;  then  eject  all  the  citrate  solution  if  less  than 
5  c.c.  blood  are  to  be  withdrawn ;  if  more  than  5  c.c.  are 
required  retain  about  half  a  cubic  centimetre  of  the  fluid 
in  the  syringe.     This  prevents  coagulation  of  the  blood. 

The  sodium  citrate  solution  is  prepared  by  dissolving : 

Sodium  citrate 10  gramme. 

Sodium  chloride.    .-.    .    .    .        0.7 5  grammes. 
In  distilled  water 100  c.c. 

Sterilise  by  boiling. 

2.  Prepare  the  animal  as  for  intravenous  inoculation 
(see  page  363)  and  introduce  the  syringe  needle  into  the 
lumen  of  the  selected  vein. 

3.  Slowly  withdraw  the  piston  of  the  syringe.     When 
sufficient  blood  has  been  collected  direct  the  assistant 
to  release  the  proximal  compression  of  the  vein;  and 
withdraw  the  needle. 

4.  Remove  the  needle  from  the  nozzle  of  the  syringe 
and  deliver  the  citrated  blood  into  a  small  Ehlenmeyer 
flask  containing  about  250  c.c.  of  nutrient  broth. 

5.  Label,  incubate  and  examine  daily  until  growth 
occurs  or  until  the  expiration  of  ten  days. 

c.  The  serological  examination  of  the  blood  is  directed 
to  the  demonstration  of  the  presence  of  certain  specific 
antibodies  in  the  sera  of  experimentally  infected  an- 
imals, and  within  certain  limits  to  an  estimation  of 
their  amounts. 

The  chief  of  these  bodies  are : 

Antitoxin. 

Agglutinin. 

Precipitin. 

Opsonin. 

Immune  body  or  Bacteriolysin. 

None  of  these  substances  are  capable  of  isolation  in 


COLLECTION    OF    SERUM  379 

a  state  of  purity  apart  from  the  blood  serum,  conse- 
quently special  methods  have  been  elaborated  to  per- 
mit of  their  recognition.  In  every  instance  the  be- 
haviour of  serum  from  the  experimental  animal, 
which  may  be  termed  "specific"  serum,  is  studied  in 
comparison  with  that  of  serum  from  an  uninoculated 
animal  of  the  same  species,  and  which  is  termed  "  nor- 
mal" serum.  In  view  of  minor  differences  in  constitu- 
tion exhibited  by  the  serum  of  various  individuals 
of  the  same  series,  it  is  usual  to  employ  a  mixture  of 
sera  obtained  from  several  different  normal  animals 
of  the  same  species  as  the  inoculated  animal,  under  the 
term  "pooled  serum."  The  method  of  collecting  blood 
(e.  g.,  from  the  rabbit)  for  serological  tests  is  as  follows : 

Collection  of  Serum. 

Apparatus  required: 

Razor. 

Liquid  soap. 

Cotton-wool. 

Lysol  2  per  cent,  solution,  in  drop  bottle. 

Ether  in  diop  bottle. 

Flat  Hagedorn  needles. 

Blood  pipettes  (Fig.  16,  page  12). 

Centrifugal  machine. 

Centrifuge  tubes. 

Glass  cutting  knife. 

Bunsen  flame. 

Writing  diamond  or  grease  pencil. 

METHOD. — 

1.  Shave  the  dorsal  surface  of  the  ear  over  the 
course  of  the  posterior  auricular  vein  (see  Fig.  192). 

2 .  Sterilise  the  skin  by  washing  with  lysol. 

The  lysol  should  be  applied  with  sterile  cotton-wool 
and  the  ear  vigourously  rubbed,  not  only  to  remove 
superficial  scales  of  epithelium,  but  also  to  render  the 
ear  hyperaemic  and  the  vein  prominent. 

3.  Remove  the  lysol  with  ether  dropped  from  a 
drop  bottle,  and  allow  the  ether  to  evaporate. 


380  EXPERIMENTAL   INFECTIONS    DURING   LIFE 

4.  Puncture  the  vein  with  a  sterile  Hagedorn  needle. 

5.  Take  a  small  blood-collecting  pipette  (Fig.    161) 
and  hold  it  at  an  angle  to  the  ear,  one  end  touching  the 
issuing  drop  of  blood,  the  other  depressed. 

The  blood  will  now  enter  the  pipette  at  first  by 
capillarity;  afterward  gravity  will  also  come  into 
play  and  the  pipette  can  be  two-thirds  filled  without 
difficulty. 

6.  Hold  the  tube  by  the  end  containing  the  blood, 
the  clean  end  pointing  obliquely  upward — warm  this 


FIG.  192. — Collecting  blood  from  rabbit. 

end  at  the  bunsen  flame  to  expel  some  of  the  contained 
air;  then  seal  the  clean  point  in  the  flame. 

7.  Place  the  pipette  down  on  a  cool  surface  (e.  g.,  a 
glass  slide) .     The  rapid  cooling  of  the  air  in  the  clean 
end  of  the  pipette  creates  a  negative  pressure,  and  the 
blood  is  sucked  back  into  the  pipette,   leaving  the 
soiled  end  free  from  blood.     Seal  this  end  in  the  bunsen 
flame. 

8.  Mark  the  distinctive  title  of  the  specimen  (e.  g., 
animal's  number)   upon  the  pipette  with  a  writing 
diamond  or  grease  pencil. 

9.  When  the  sealed  ends  are  cold  and  the  blood  has 
clotted,  place  the  pipette  on  the  centrifuge,  clean  end. 
downward ;  counterpoise  and  centrifugalise  thoroughly. 
On  removing  the  pipette  from  the  centrifuge,  the  red 
cells  will  be  collected  in  a  firm  mass  at  one  end,  and 
above  them  will  appear  the  clear  serum. 


DILUTION    OF    THE    SPECIFIC    SERUM  381 

10.  By  marking  the  blood  pipette  above  the  level 
of  the  serum  with  the  glass  cutting  knife  and  snapping 
the  tube  at  that  point,  the  blood-serum  becomes 
readily  accessible  for  testing  purposes. 

If  larger  quantities  of  blood  are  required,  the  animal, 
after  puncturing  the  vein,  should  be  inverted,  an 
assistant  holding  it  up  by  the  legs.  Blood  to  the 
volume  of  several  cubic  centimetres  will  now  drop  from 
the  punctured  vein,  and  should  be  caught  in  a  tapering 
centrifuge  tube,  the  tube  transferred  to  the  incubator 
at  37°  C.  for  two  hours,  then  placed  in  the  centrifugal 
machine,  counterpoised  and  centrifugalised  thoroughly. 
The  three  most  important  of  the  antibodies  referred  to 
which  can  be  demonstrated  with  a  certain  amount  of 
facility  are  agglutinin,  opsonin  and  bacteriolysin ;  and 
the  methods  of  testing  for  these  bodies  will  now  be 
considered. 

AGGLUTININ. 

Agglutinin  is  the  name  given  to  a  substance  present 
in  the  blood-serum  of  an  animal  that  has  successfully 
resisted  inoculation  with  a  certain  micro-organism. 
This  substance  possesses  the  power  of  collecting 
together  in  clumps  and  masses,  or  agglutinating  watery 
suspensions  of  that  particular  microbe. 

Dilution  of  the  Specific  Serum : 

Apparatus  required: 

Sterile  gx  aduated  capillary  pipettes  to  contain  ice.  mm.  (Fig.  17). 

Sterile  graduated  capillary  pipettes  to  contain  90  c.  mm.  (Fig.  17). 

Small  sterile  test-tubes  5X0.5  cm. 

Normal  saline  solution  in  flask  or  test-tube. 

Pipette  of  specific  serum. 

Glass  cutting  knife,  or  three-square  file. 

Glass  capsule,  nearly  full  of  dry  silver  sand,  or  roll  of  plasticine. 

Grease  pencil. 


382         EXPERIMENTAL  INFECTIONS  DURING  LIFE 

METHOD.— 

i.'  Take  three  sterile  test-tubes  and  number  them  i, 
2  and  3. 

2.  Pipette  o..9  c.c.  sterile  normal  saline  solution  into 
each  tube,  and  stand  tubes  upright  in  the  sand  in  the 
capsule,  or  in  the  plasticine  block. 

3.  Make  a  scratch  with  the  glass  cutting  knife  on  the 
blood  pipette  above  the  upper  level  of  the  clear  serum, 
and  snap  off  and  discard  the  empty  portion  of  the  tube. 

4.  Remove  o.i   c.c.  of  the  serum  from  the  blood 
pipette  tube,  and  mix  it  thoroughly  with  the  fluid  in 
tube  No.  i ;  and  label  s.s.,  (specific  serum),  10  per  cent. 

5.  Remove  o.i  c.c.  of  the  solution  from  tube  No.  i 
by  means  of  a  fresh  pipette,  and  mix  it  with  the  con- 
tents of  tube  No.  2 ;  and  label  s.s.,  i  per  cent. 

6.  Remove  o.i  c.c.  of  the  solution  from  tube  No.  2 
by  means  of  a  fresh  pipette,  and  mix  it  with  the  con- 
tents of  tube  No.  3 ;  and  label  s.s.,  o.i  per  cent. 

When  the  yield  of  serum  from  the  specimen  of  blood 
which  has  been  collected,  or  is  available,  is  small,  the 
above  method  of  diluting  is  not  practicable,  and  the 
dilution  should  be  carried  out  by  Wright's  method 
in  a  capillary  teat  pipette. 

Dilution  of  Serum  by  Means  of  a  Teat  Pipette. 

Materials  required: 

Blood  pipette  containing  sample  of  specific  serum  after  centrif- 
tigalisation. 

Capsule  of  diluting  fluid — normal  saline  solution, 

Supply  of  Pasteur  pipettes  (Fig.  130). 

India-rubber  teats. 

Small  test-tubes. 

A  block  of  plasticine  to  act  as  a  test-tube  stand. 

Grease  pencil. 

METHOD: 

i .  Mark  three  small  test-tubes  10  per  cent.,  i  per  cent, 
and  o.i  per  cent,  respectively,  and  stand  them  upright 
in  the  plasticine  block. 


DILUTION  OF  SERUM  BY  MEANS  OF  A  TEAT  PIPETTE       383 

2.  Take  a  Pasteur  pipette,  nick  the  capillary  stem 
just  above  the  sealed  end  with  a  glass  cutting  knife,  and 
snap  off  the  sealed  end  with  a  quick  movement  so  that 
the  fracture  is  clean  cut  and  at  right  angles  to  the  long 
axis  of  the  capillary  stem — cut  "square",  in  fact. 
Prepare  several,  say  a  dozen,  in  this  manner. 

.3.  Fit  a  rubber  teat  to  the  barrel  of  each  of  the 
pipettes. 

4.  Make  a  mark  with  the  grease  pencil  on  the  stem 
of  one  of  the  pipettes  about  2  or  3  cm.  from  the  open 
extremity. 


FIG.   193. — Filling  the  capillary  teat  pipette. 

5.  Compress  the  teat  between  the  ringer  and  thumb 
(Fig.  193)  to  such  an  extent  as  to  drive  out  the  greater 
part  of  the  contained  air. 

6.  Maintaining  the  pressure  on  the  teat  pass  the 
stem    of   the   pipette   into   the   capsule   holding   the 
saline  solution,  until  the  open  end  of  the  pipette  is 
below  the  level  of  the  fluid. 

7.  Now   cautiously  relax  the  pressure  on  the  teat 
and  let  the  fluid  enter  the  pipette  and  rise  in  the  stem 
until  it  reaches  the  level  of  the  grease  pencil  mark.     As 
soon  as  this  point  is  reached,  check  the  movement  of 


384  EXPERIMENTAL   INFECTIONS    DURING    LIFE 

the  column  of  fluid  by  maintaining  the  pressure  on  the 
teat,  neither  relaxing  nor  increasing  it. 

8.  Withdraw  the  point  of  the  pipette  clear  of  the 
fluid,  and  again  relax  the  pressure  on  the  teat  very 
slightly.     The  column  of  saline  solution  rises  higher  in 
the  stem,  and  a  column  of  air  will  now  enter  the  pipette 
and  serve  as  an  index  to  separate  the  first  volume  of 
fluid  drawn  into  the  stem  from  the  next  succeeding  one. 

9.  Again  introduce  the  end  of  the  pipette  into  the 
fluid  and  draw  up  a  second  volume  of  saline  to  the  level 
of  the  grease  pencil  mark,  and  follow  this  with  a  second 
air  index. 

10.  In  like  manner  take  up  seven  more  equal  volumes 
of   saline   solution   and   their   following   air   bubbles. 
There  are  now  nine  equal  volumes  of  normal  saline  in 
the  pipette. 

1 1 .  Now  pass  the  point  of  the  pipette  into  the  blood 
tube  and  dip  the  open  end  below  the  surface  of  the 
serum.     Proceeding  as   before,    aspirate  a  volume  of 
serum  into  the  capillary  stem  up  to  the  level  of  the 
pencil  mark. 

12.  Eject  the  contents  of  the  pipette  into  the  small 
tube  marked  10  per  cent,  by  compressing  the  rubber 
teat  between  thumb  and  ringer. 

13.  Mix  the  one  volume  of  serum  with  the  nine 
volumes   of   saline   solution   very  thoroughly   by  re- 
peatedly drawing  up  the  whole  of  the  fluid  into  the 
pipette  and  driving  it  out  again  into  the  test-tube. 

14.  Now  take  a  clean  pipette  and  proceed  precisely 
as  before,  4  to  10. 

15.  Having  aspirated  nine  equal  volumes  of  saline 
into  this  second  pipette,   now  take  up  one  similar 
volume  of  the  fluid  in  the  "  10  per  cent,  tube." 

1 6.  Eject  the  contents  of  this  pipette  into  the  second 
tube  marked  i  per  cent,  and  mix  thoroughly  as  before. 

17.  In  similar  fashion  make  the  o.i  per  cent,  solution 
and  transfer  to  the  third  tube. 


THE    MICROSCOPICAL   REACTION  385 

18.  Further  dilutions  in  multiples  of  ten  can  be  pre- 
pared in  the  same  way,  and  by  varying  the  number 
of  volumes  of  diluting  fluid  or  serum  any  required 
dilution  can  be  made  (see  Appendix,  Dilution  Tables) . 

NOTE. — The  saline  diluting  fluid  must  always  be  taken  into  the 
pipette  first,  otherwise  if  the  serum,  contains  a  very  large  amount 
of  agglutinin  the  traces  of  this  serum  added  to  the  saline  solu- 
tion may  be  sufficient  to  entirely  vitiate  the  subsequent  observa- 
tions— whilst  if  more  than  one  sample  of  serum  is  diluted  from  the 
same  saline  solution  serious  errors  may  be  introduced  into  the 
experiments. 

The  Microscopical  Reaction : 

Apparatus  Required: 

Five  hanging-drop  slides  (or  preferably  two  slides,  with  two  cells 
mounted  side  by  side  on  each  (Fig.  62,  a),  and  one  slide  with  one 
cell  only. 

Vaseline. 

Cover-slips. 

Platinum  loop. 

Grease  pencil. 

Eighteen  to  twenty-four-hour-old  bouillon  cultivation  of  the 
organism  to  be  tested  (e.g.,  Bacillus  typhi  abdominalis) 

Pipette  end  with  the  remainder  of  the  specific  serum  labelled  s.s. 

Tubes  containing  the  three  solutions  of  the  specific  serum,  10, 
i,  and  o.i  per  cent,  respectively. 

Pipette  end  with  pooled  normal  serum  labelled  p.s. 

METHOD. — 

i.  Make  five  hanging-drop  preparations,  thus: 

(a)  One  loopful  of  bouillon  cultivation  +  one  loopful 
pooled  serum;  label  "Control." 

(6)  One  loopful  culture  +  one  loopful  undiluted 
specific  serum;  label  50  per  cent. 

Mount  these  two  cover-slips  on  a  double-celled  slide. 

(c)  One   loopful  bouillon  culture   -f  one  loopful  10 
per  cent,  serum;  label  5  per  cent. 

Mount    this    on    single-cell    slide. 

(d)  One  loopful  bouillon  culture  +  one  loopful  i  per 
cent,  serum;  label  0.5  per  cent. 

(e)  One  loopful  bouillon  culture  -f  one  loopful  o.i  per 
cent,  serum;  label  0.05  per  cent. 

25 


386  EXPERIMENTAL   INFECTIONS    DURING    LIFE 

Mount  these  two  cover-slips  on  a  double-celled  slide. 

2.  Note  the  time:  Examine  the  control  to  determine 
that  the  bacilli  are  motile  and  uniformly  scattered  over 
the  field — not  collected  into  masses. 

3.  Next  examine  the  50  per  cent,  serum  preparation. 
If  agglutinin  is  present   and   the  test  is  giving  a 

positive  reaction,  the  bacilli  will  be  collected  in  large 
clumps. 

If  the  test  is  giving  a  negative  reaction,  the  bacilli 
may  be  collected  in  large  clumps  owing  to  the  viscosity 
of  the  concentrated  serum. 

4.  Observe  the  5  per  cent,  preparation  microscopic- 
ally. 

If  the  bacilli  are  aggregated  into  clumps,  positive 
reaction. 

If  the  bacilli  are  not  aggregated  into  clumps,  observe 
until  thirty  minutes  from  the  time  of  preparation  before 
recording  a  negative  reaction. 

5.  Examine  the  0.5  and  0.05  per  cent,  preparations. 
These  may  or  may  not  show  agglutination  when  the 

result  of  the  examination  of  the  5  per  cent,  preparation 
is  positive,  according  to  the  potency  of  the  specific 
serum ;  and  by  the  examination  of  a  series  of  dilutions 
a  quantitative  comparison  of  the  valency  of  specific 
sera  from  different  sources,  or  of  serum  from  the  same 
animal  at  different  periods  during  the  course  of  active 
immunisation  may  be  obtained. 

NOTE. — The  graduated  pipettes  supplied  with  Thoma's  hsema- 
tocytometer  (intended  for  the  collection  of  the  specimen  of  blood 
required  for  the  enumeration  of  leucocytes),  giving  a  dilution  of 
i  in  10 — i.  e.,  10  per  cent. — may  be  substituted  for  the  graduated 
capillary  pipettes  referred  to  above,  if  the  vessel  in  which  the 
serum  has  been  separated  is  of  sufficiently  large  diameter  to  per- 
mit of  their  use. 

The  Macroscopical  Reaction : 

Sterile  graduated  capillary  pipettes  to  contain  90  c.  mm. 
Eighteen   to  twenty-four-hours-old  bouillon  cultivation  of  the 
organism  to  be  tested. 


THE    MACROSCOPICAL    REACTION  387 

Three  test-tubes  containing  the  10,  i,  and  o.i  per  cent,  solutions 
of  specific  serum  (about  90  c.  mm.  remaining  in  each) . 
Tube  containing  50  per  cent,  solution  of  pooled  serum. 
Sedimentation  pipettes  (vide  page  1 7)  or  teat  pipettes. 

METHOD.— 

1.  Pipette  90  c.  mm.  of  the  bouillon  culture  into  each 
of  the  tubes   containing  the  diluted  serum;  and  the 
same  quanitity  into  the  tube  containing  the  pooled 
serum. 

2.  Fill  a  sedimentation  tube  (by  aspirating)  or  a  teat 
pipette  from  the  contents  of  each  tube.     Seal  off  the 
lower  ends  of  the  sedimentation  tubes  in  the  Bunsen 
flame. 

3 .  Label  each  tube  with  the  dilution  of  serum  that  it 
contains- — viz.,  5,  0.5,  and  0.05  per  cent. 

4.  Place  the  pipettes  in  a  vertical  position,   in  a 
beaker,  in  the  incubator  at  37°  C.,  for  one  or  two  hours. 

5.  Observe  the  granular  precipitate  which  is  thrown 
down  when  the  reaction  is  positive,  and  the  uniform 
turbidity  of  the  negative  reaction  as  compared  with  the 
appearances  in  the  control  pooled  serum. 

OPSONIN. 

Opsonin  is  the  term  applied  by  Wright  to  a  substance, 
present  in  the  serum  of  an  inoculated  animal,  which  is 
able  to  act  upon  or  sensitise  bacteria  of  the  species 
originally  injected,  so  as  to  render  them  an  easy  prey 
to  the  phagocytic  activity  of  polymorphonuclear  leuco- 
cytes. In  the  method  for  demonstrating  opsonin  about 
to  be  described,  a  comparison  is  made  between  the 
.opsonic  " power"  of  the  pooled  serum  and  the  specific 
serum. 


Apparatus: 

Small  centrifuge  and  tubes  for  same  (made  from  the  barrels 
of  broken  capillary  pipettes  by  sealing  the  conical  ends  in  the 
bunsen  flame). 

Capillary  Pasteur  pipettes. 

India-rubber  teats. 


388  EXPERIMENTAL    INFECTIONS    DURING   LIFE 

Grease  pencil. 

Bunsen  burner  with  peep  flame. 

Electrical  signal  clock  (see  page  39)  stop  watch,  or  watch. 

Rectangular  glass  box  or  tray  to  hold  pipettes. 

Incubator  regulated  at  37°  C. 

3X1  slides. 

Piece  of  light  rubber  tubing. 

Retangular  block  of  plasticine. 

Flask  of  normal  saline  solution. 

Flask  of  sodium  citrate  (1.5  per  cent.)  in  normal  saline  solution. 
Materials  required,  and  their  preparation: 

Small  tube  of  "washed  cells"  (red  blood  discs  and  leucocytes); 
human  cells  are  used  in  estimating  the  opsoiiising  power  of  the 
serum  of  experimental  animals. 

Small  tube  of  emulsion  of  bacteria  of  the  species  responsible 
for  the  infection  of  the  experimental  animal. 

Blood  pipette  containing  specific  serum. 

Blood  pipette  containing  "pooled"  serum. 

Washed  Cells. — 

1.  Take  a  small  centrifuge  tube  and  half  fill  it  with 
sodium  citrate  solution.     Mark  with  the  grease  pencil 
the  upper  limit  of  the  fluid. 

2 .  Cleanse  the  skin  of  the  distal  phalanx  of  the  second 
finger  of  the  left  hand  above  the  root  of  the  nail  with 
lint  and  ether.     Wind  the  rubber  tubing  tightly  round 
the  second  phalanx ;  puncture  with  a  sterile  Hagedorn 
needle  through  the  cleansed  area  of  skin. 

3 .  Take  up  a  sufficiency  of  the  issuing  blood  (more  or 
less  according  to  the  number  of  tests  to  be  performed) 
with  a  teat  pipette,  transfer  it  to  the  tube  of  citrate 
solution  and  mix  thoroughly.     Make  a  second  mark  on 
the  tube  at  the  upper  level  of  the  mixed  citrate  solution 
and  blood. 

4.  Place  the   tube  in   the   centrifuge,    counterpoise 
accurately  and  centrifugalise  until  the  blood  cells  are 
thrown  down  in  a  compact  mass  occupying  approxi- 
mately the  same  volume  as  is  included  between  the 
two  pencil  marks. 

The  column  of  fluid  in  the  tube  now  shows  clear 
supernatant  fluid  (citrate  solution  and  blood  plasma) 


ESTIMATION    OF    OPSONIN  389 

separated  from  the  sharp  cut  upper  surface  of  the  red 
deposit  of  corpuscles  by  a  narrow  greyish  layer  of 
leucocytes. 

5.  Remove  the  supernatant  column  of  citrate  solution 
by  means  of  a  teat  pipette,  fill  normal  saline  solution 
into  the  tube  up  to  the  upper  pencil  mark,  and  dis- 
tribute the  blood  cells  throughout  the  saline  by  means 
of  the  teat  pipette.     Centrifugalise  as  before. 

6.  Again  remove  the  supernatant  fluid  and  fill  in  a 
fresh  supply  of  saline  solution  and  centrifugalis  once 
more. 

7.  Remove  the  supernatant  saline  solution  as  nearly 
down  to  the  level  of  the  leucocytes  as  can  be  safely 
done  without  removing  any  of  the  leucocytes. 

8.  Next  distribute  the  leucocytes  evenly  throughout 
the  mass  of  red  cells  by  rotating  the  tube  between  the 
palms  of  the  hands — just  as  is  done  with  a  tube  of 
liquefied  medium  prior  to  pouring  a  plate. 

9.  Set  the  tube  upright  in  the  plasticine  block  neal 
to  one  end. 

Bacterial  Emulsion. — 

1.  Take  an  18-  to  24-hour  culture  of  the  required 
bacterium  (e.  g.,  Diplococcus  pneumonias)  grown  upon 
sloped  blood  agar  at  37°  C.     Pour  over  the  surface  of 
the  medium  some  5  c.c.  of  normal  saline  solution. 

2.  With  a  platinum  loop  emulsify  the  growth  from 
the  surface  of  the  medium  as  evenly  as  possible  in  the 
saline  solution. 

3.  Allow  the  tube  to  stand  for  a  few  minutes  so  that 
the  large  masses  of  growth  may  settle  down ;  transfer  the 
upper  portion  of  the  saline  suspension  to  a  centrifuge 
tube  and  centrifugalise  thoroughly. 

4.  Examine  a  drop  of  the  supernatant  opalescent 
emulsion  microscopically  to  determine  its  freedom  from 
clumps  and  masses.     If  unsatisfactory  prepare  another 
emulsion,  this  time  scraping  up  the  surface  growth  with 


390 


EXPERIMENTAL    INFECTIONS    DURING   LIFE 


a  platinum  spatula,  transferring  it  to  an  agate  mortar 
and  grinding  it  up  with  successive  small  quantities  of 
normal  saline.  If  satisfactory  insert  the  tube  in  the 
plasticine  block  next  to  that  containing  the  washed  cells. 

Specific  Serum. — 
Pooled  Serum. — 

These  sera  are  collected  and  treat- 
ed as  already  described   (see  page 
379),  and  the  portions  of  the  blood 
pipettes   containing    them    are    ar- 
.T94-  Plasticine  ranged   in   the  remaining  space  in 

block  with  materials  ar-  °  . 

ranged  for  opsonin  esti-    plasticine  block. 

mations-  The  plasticine  block  now  presents 

the  appearances  shown  in  Fig.  194. 

METHOD  FOR  DETERMINING  THE  OPSONIC  INDEX. — 

1.  Take  a  capillary  pipette  fitted  with  a  teat,  cut  the 
distal  end  square  and  make  a  pencil  mark  about  2  cm. 
from  the  end. 

2.  Aspirate  into  the  pipette  one  volume  of  washed 
cells,  air  index,  one  volume  of  bacterial  emulsion,  air 
index,  and  one  volume  of  specific  serum  (see  Fig.  195). 


Serum 


Bacterial       Washed  cells 
emulsion 

FIG.   195,     Opsonin  pipette. 


3.  Mix  thoroughly  on  a  3  by  i  slide  by  compressing 
the  teat  and  ejecting  the  contents  of  the  pipette  on 
to  the  surface  of  the  slide,  relaxing  the  pressure  and 
so  drawing  the  fluid  up  into  the  pipette  again.     These 
two  processes  should  be  repeated  several  times ;  finally 
take  up  the  mixture  in  an  unbroken  column  to  the  cen- 
tral portion  of  the  capillary  stem. 

4.  Seal  the  point  of  the  pipette  in  the  peep  flame  of 
the  bunsen  burner  and  remove  teat. 


ESTIMATION  OF  OPSONIN  391 

5.  Mark  the  pipette  (with  the  grease  pencil)  with  the 
distinctive  number  of  the  serum  and  place  it  in  the 
glass  box  or  tray. 

6.  Take  another  similarly  prepared  pipette  and  aspi- 
rate into  it  equal  volumes  of  washed  cells,  bacterial 
emulsion  and  pooled  serum.     Treat  precisely  as  in  3 
and  4,  label  it  "control"  or  "N.S."    (normal  serum) 
and  place  in  the  box  by  the  side  of  the  specific  serum 
preparation. 

7.  Place  the  box  with  the  pipettes  in  the  incubator 
and  set  the  signal  clock  to  ring  at  15  minutes  (or  start 
the  stop  watch). 

6.  At  the  expiration  of  the  incubation  time  remove 
the  pipettes  from  the  incubator. 

9.  Cut  off  the  sealed  end  of  the  specific  serum  prepa- 
ration.    Mix  its  contents  thoroughly  as  in  step  3,  and 
then  divide  the  mixture  between  two  3  by  i  slips  and 
carefully  spread  a  blood  film  (vide  page  376)  on  each 
in  such  a  way  that  only  one-half  of  the  surface  of  each 
slide  is  covered  with  blood — the  free  edge  of  the  blood 
film  approximating  to  the  longitudinal  axis  of  the  slide. 

Allow  films  to  dry  and  label  the  slides  with  writing 
diamond. 

10.  Treat   the   contents  of  the  control  pipette  in 
similar  fashion. 

11.  Select  the  better  film  from  each  pair  for  fixing 
and  staining. 

12.  Fixing  and  staining  must  be  carried  out  under 
strictly  comparable  conditions,   and  to  this  end  the 
slides  are  best  handled  by  placing  in  a  glass  staining 
rack  which  can  be  lowered  in  turn  into  each  of  a  series 
of  glass  troughs  containing  the  various  reagents  (Fig. 
196).     Place  the  rack  in  the  first  trough  which  contains 
the  alcoholic  solution  of  Leishman's  stain  for  two  min- 
utes to  fix. 

Transfer  to  the  second  trough  containing  the  diluted 
stain  for  ten  minutes. 


392  EXPERIMENTAL   INFECTIONS    DURING   LIFE 

Transfer  to  the  third  trough  containing  distilled 
water,  and  holding  the  trough  over  a  sink,  run  in  a 
stream  of  distilled  water  until  washing  is  complete. 
Remove  slides  from  the  rack  and  dry. 

Leishman's  stain  is  the  best  for  routine  work  for  all 
bacteria  other  than  B.  tuberculosis.  Films  containing 
tubercle  bacilli  must  of  course  be  stained  by  the  Ziehl 
Neelsen  method. 


FIG.  196.     Glass  staining  trough  for  blood  films. 

1 3 .  Examine  specific  serum  slide  microscopically  with 
i/ 12  inch  oil  immersion.     Find  the  edge  of  the  blood 
film — along  this  the  bulk  of  the  leucocytes  will  be 
collected.     Starting  at  one  end  of  the  film  move  the 
slide  slowly  across  the  microscope  stage  and  as  each 
leucocyte  comes  into  view  count  and  record  the  number 
of  ingested  bacteria.     The  sum  of  the  contents  of  the 
first  50  consecutive  polymorphonuclears  that  are  en- 
countered is  marked  down.     (The  average  number  of 
bacilli  ingested  per  leucocyte  =  the  " pkagocytic  index.") 

14.  In  precisely  similar  manner  enumerate  the  bac- 
teria present  in  the  first  50  cells  of  the  control  prepa- 
ration.    This  number  is  recorded  as  the  denominator 


COMPLEMENT    FIXATION  393 

of  a  vulgar  fraction  of  which  the  numerator  is  the 
number  recorded  for  the  specific  serum.  This  fraction, 
expressed  as  a  percentage  of  unity  =  the  opsonic  index. 

IMMUNE  BODY. 

Immune  body  or  amboceptor  is  the  name  given  to  a 
substance  present  in  the  serum  of  an  infected  animal 
that  has  successfully  resisted  inoculation  with  some  par- 
ticular micro-organism,  and  which  possesses  the  power 
of  linking  the  complement  normally  present  in  the 
serum  to  bacteria  of  the  species  used  as  antigen  in  such 
a  manner  that  the  micro-organisms  are  rendered  in- 
nocuous, and  ultimately  destroyed.  The  presence  of 
the  immune  body  in  the  serum  can  be  demonstrated 
in  vitro  by  the  reaction  elaborated  by  Bordet  and  Gen- 
gou,  known  as  the  complement  fixation  test,  the  exist- 
ence or  the  absence  of  the  phenomenon  of  complement 
fixation  being  rendered  obvious  macroscopically  by  the 
absence  or  presence  of  haemolysis  on  the  subsequent 
addition  of  ' 'sensitised"  red  blood  corpuscles,  (e.g.,  a 
mixture  of  erythrocyte  solution  and  the  appropriate 
hasmolysin — two  of  the  three  essentials  in  the  hsemoly- 
tic  system,  vide  page  326). 

Apparatus  Required: 

Sterile  pipettes  i  c.c.,  (graduated  in  tenths). 

16X2  cm.  test-tubes. 

9X1  cm.  test-tubes. 

Test-tube  racks  for  each  size  of  test-tube. 
Reagents  Required: 

Normal  saline  solution. 

Erythrocyte  solution  (human  red  cells,  page  329)  =E. 

Haemolytic  serum  (for  human  cells)  =  H.S. 

Complement  (fresh  guinea-pig  serum)  =  C. 

Specific  serum  from  inoculated  animal,  inactivated  =  S.S. 

Control  pooled  serum  from  normal  animals  of  same  species, 
inactivated  =  P.S. 

Antigen  (cultivation  upon  solid  medium  of  the  organism  (e.g.,  B. 
typhosus)  which  has  already  served  as  antigen  in  the  inoculation 
of  the  experimental  animal)  =A 


394  EXPERIMENTAL   INFECTIONS    DURING   LIFE 

To  prepare  the  antigen  for  use,  emulsify  the  whole  of 
the  bacterial  growth  in  5  c.c.  normal  saline  solution. 

Shake  the  emulsion  in  a  test-tube  with  some  sterilised 
glass  beads  to  ensure  a  homogenous  emulsion,  and  ster- 
ilise by  heating  to  60°  C.  in  a  water-bath  for  one  hour. 

METHOD. — • 

1.  Take  five  small  test-tubes,  and  number  them  i  to  5 
with  a  grease  pencil. 

2.  Into  tubes  Nos.  i,  3,  4  and  5  pipette  o.i  c.c.  of 
complement. 

3.  Into  tubes  Nos.  i  and  2  pipette  0.2  c.c.  of  the 
serum  to  be  tested. 

4.  Into  tube  No.  4  pipette  0.2  c.c.  of  control  serum. 

5.  Into  tubes  Nos.  i,  2,  3  and  4  pipette  i  c.c.  of  the 
bacterial  emulsion  which  forms  the  antigen. 

6.  Place  the  whole  set  of  tubes  in  the  incubator  at 
37°  C.  for  a  period  of  one  hour. 

7.  Remove  the  tubes  from  the  incubator  and  pipette 
i  c.c.  erythrocyte  solution  and  4  minimal  hasmolytic 
doses  of  the  corresponding  haemolysin  into  each  tube. 

8.  Mix  thoroughly  and  return  the  tubes  to  the  incu- 
bator at  3  7°  C.  for  further  period  of  one  hour. 

9.  At  the  expiration  of  that  time  transfer  the  tubes 
to  the  ice  chest,  and  allow  them  to  stand  for  three 
hours. 

10.  Examine  the  tubes. 

Tubes  3,  4  and  5  should  show  complete  haemolysis; 
tube  2  should  give  no  evidence  whatever  of  haemolysis. 

These  tubes  form  the  controls  to  the  first  tube,  which 
contains  the  serum  to  be  tested. 

In  tube  No.  i  the  absence  of  haemolysis  would  indicate 
the  presence  in  the  serum  of  the  inoculated  animal  of  a 
specific  antibody  to  the  micro-organism  used  in  the 
inoculations;  since  it  shows  that  the  complement  has 
been  bound  by  the  immune  body  to  the  bacterial 
antigen,  and  none  has  been  left  free  to  enter  into  the 


COMPLEMENT    FIXATION  395 

hasmolytic  system ;  on  the  other  hand  the  presence  of 
haemolysis  would  show  that  no  appreciable  amount  of 
antibody  has  yet  been  formed  in  response  to  the  inocu- 
lations. In  other  words,  there  is  an  absence  of  infec- 
tion, since  the  complement  remained  unfixed  at  the 
time  of  the  addition  of  the  erythrocyte  solution  and 
haemolytic  serum,  and  was  ready  to  combine  with  those 
reagents  to  complete  the  haemolytic  system. 

The  method  may  be  shown  diagramatically  as  under 
using  the  symbols  already  indicated  • 

Test-tubes. 


© 

O.I    C.C.  C. 
O.2    C.C.    S.S. 

A. 

© 

® 

o.i  c.c.  C. 
A. 

©                   © 
o.i  c.c.  C.        o.i  c.c.  C. 

0.2    C.C.   P.S  

A.                 

0.2    C.C.    S.S. 

A. 

Incubate  at 

3  7°  C.  for 

one  hour. 

i  c.c.  E. 
H.S.4 

i.  c.c.  E. 
H.S.4 

i  c.c.  E. 
H.S.4 

i  c.c.  E.            i  c.c.  E. 
H.S.4                 H.S.4 

Incubate  at 

3  7°  C.  for 

one  hour. 

(?) 

No  haemolysis. 

Haemolysis. 

NOTE. — It  is  sometimes  more  convenient  to  sensitise  the  erythro- 
cytes  just  before  they  are  needed.  This  is  done  forty-five  minutes 
after  the  experiment  has  been  started  (page  394,  step  6),  that  is  to 
say,  before  the  completion  of  the  first  period  of  incubation,  thus: 

1.  Measure  out  into  a  sterile  test-tube   (or  flask)'  five  c.c.  of 
erythrocyte  solution. 

2.  Measure  out  twenty  minimal  haemolytic  doses  of  haemolysin, 
add  to  the  erythrocyte  solution  on  the  test-tube. 

3.  Allow  the  erythrocyte  and  haemolysin  to  remain  in  contact  for 
fifteen  minutes  at  room  temperature.    The  red  cells  are  then  sensi- 
tised and  ready  for  use. 

4.  When  the  tubes  are  removed  from  the  incubator  at  the  end  of 
the  first  hour  (i.e.,  step  7)   add  i  c.c.  sensitised  red  cells  to  each 
tube  by  means  of  a  graduated  pipette. 

5.  Mix  thoroughly,  return  the  tubes  to  the  incubator  at  37°  C. 
and   complete  the  experiment  as  previously  described  (steps  8 
onward) . 


XIX.    POST-MORTEM  EXAMINATIONS  OF 
EXPERIMENTAL  ANIMALS. 

THE  post-mortem  examination  should  be  carried  out 
as  soon  as  possible  after  the  death  of  the  animal,  for  it 
must  be  remembered  that  even  in  cold  weather  the 
tissues  are  rapidly  invaded  by  numerous  bacteria 
derived  from  the  alimentary  tract  or  the  cavities  of 
the  body,  and  from  external  sources. 

The  following  outlines  refer  to  a  complete  and  ex- 
haustive necropsy,  and  in  routine  work  the  examination 
will  rarely  need  to  be  carried  out  in  its  entirety. 

NOTE. — Throughout  the  autopsy  the  searing  irons  must  be 
freely  employed,  and  it  must  be  recollected  that  one  instrument 
is  only  to  be  employed  to  seize  or  cut  one  structure.  This  done, 
it  must  be  regarded  as  contaminated  and  a  fresh  instrument  taken 
for  the  next  step. 

Apparatus  Required : 

Water  steriliser. 

[  Scalpels. 

Surgical  instruments:     1  ^cissors- 
]  Forceps. 

[  Bone  forceps. 

Spear-headed  platinum  spatula  (Fig.  199). 
Searing  irons  (Fig.  198). 
Tubes  of  media — bouillon  and  sloped  agar. 

Surface  plates  in  petri  dishes  (of  agar  or  one  of  its  derivatives) . 
Platinum  loop. 
Aluminium  ' '  spreader. ' ' 
Grease  pencil. 

Sterile  capillary  pipettes  (Fig.  13,  a). 
Sterile  glass  capsules,  large  and  small. 
Cover-slips  or  slides. 

Bottles  of  fixing  fluid  (vide  page  114)  for  pieces  of  tissue  intended 
for  sectioning. 

396 


APPARATUS    REQUIRED 


397 


1.  Place  the  various  instruments,  forceps,  scissors, 
scalpels,  etc.,  needed  for  the  autopsy  inside  the  steriliser 
and  sterilise  by  boiling  for  ten  minutes;  then  open  the 
steriliser,  raise  the  tray  from  the  interior  and  rest  it 
crosswise  on  the  edges. 

2 .  Heat  the  searing  irons  to  redness  in  a  separate  gas 
stove. 

3.  Drench  the  fur  (or  feathers)  with  lysol  solution, 
2  per  cent.     This  serves  the  twofold  purpose  of  pre- 


FIG.  197. — Apparatus  for  post-mortem  examination,  animal  on  board. 

venting  the  hairs  from  flying  about  and  entering  the 
body  cavities  during  the  autopsy,  and  of  rendering 
innocuous  any  vermin  that  may  be  present  on  the 
animal. 

4.  Examine  the  cadaver  carefully  c     Recollect  that 
laboratory  animals  are  not  always  hardy ;  death  may  be 


FIG.  198. — Searing  iron. 

due  to  exposure  to  heat  or  cold,  to  starvation  or  over- 
or  improper  feeding  or  to  the  attack  of  rats — and  not 
to  the  bacterial  infection. 

5.  Fasten  the  body  of  the  animal,  ventral  surface 
upward  (unless  there  is  some  special  reason  for  having 


398  POST-MORTEM    EXAMINATIONS 

the  dorsum  exposed) ,  out  on  a  board  by  means  of  copper 
nails  driven  through  the  extremities. 

6.  With  sterile  forceps  and  scalpel  incise  the  skin 
in  the  middle  line  from  the  top  of  the  sternum  to  the 
pubes.     Make  other  incisions  at  right  angles  to  the 
first  out  to  the  axillae  and  groins,  and  reflect  the  skin 
in  two  lateral  flaps.     (Place  the  now  infected  instru- 
ments on  the  board  by  the  side  of  the  body  or  support 
them  on  a  porcelain  knife  rest.) 

Seat  of  Inoculation. — 

7.  Inspect  the  seat  of  inoculation.     If  any  local  les- 
ion is  visible,  sear  its  exposed  surface  and  with  the  plat- 
inum loop,  remove  material  from  the  deeper  parts  to 
make  tube  and  surface  plate  cultivations  and  cover-slip 
preparations 

Collect   specimens   of   pus   or  other    exudation    in 
capillary  pipettes  for  subsequent  examination. 

8.  Inspect  the  neighbouring  lymphatic  glands  and 
endeavour  to  trace  the  path  of  the  virus. 

9.  Sear  the  whole  of  the  exposed  surface  of  the 
thorax  with  the  searing  irons. 

Pleural  Cavity.— 

10.  Divide  the  ribs  on  either  side  of  the  sternum  and 
remove  a  rectangular  portion  of  the  anterior  chest  wall 
with  sterile  scissors  and  a  fresh  pair  of  forceps,  exposing 
the  heart.     Place  the  infected  instruments  by  the  side 
of  the  first  set. 

1 1 .  Observe  the  condition  of  the  anterior  mediastinal 
glands,  the  thymus  and  the  lungs.     Collect  a  quantity 
of  pleuritic  effusion,  if  such  is  present,  in  a  pipette  for 
further  examination  later. 

1 2 .  Raise  the  pericardial  sac  in  a  fresh  pair  of  forceps 
and  burn  through  this  structure  with  a  searing  iron. 

Collect  a  sample  of  pericardial  fluid  in  a  pipette  for 
microscopical  and  cultural  examination. 


PERITONEAL    CAVITY  399 

13.  Grasp  the  apex  of  the  heart  in  the  forceps  and 
sear  the  surface  of  the  right  ventricle. 

14.  Plunge  the  open  point  of  a  capillary  pipette 
through  the  seared  area  into  the  ventricle  and  fill  with 
blood. 

Make  cultivations  and  cover-slip  preparations  of  the 
heart  blood. 

15.  Collect  a  further  sample  of  blood  or  serum  for 
subsequent  investigation  as  to  the  presence  of  anti- 
bodies. 

Peritoneal  Cavity.— 

1 6.  Sear  a  broad  track  in  the  middle  line  of  the  abdom- 
inal wall;  open  the  peritoneal  cavity  by  an  incision 
in  the  centre  of  the  seared  line.     Observe  the  condition 
of  the  omentum,  the  mesentery,  the  viscera  and  the 
peritoneal  surface  of  the  intestines. 

17.  Collect  a  specimen  of  the  peritoneal  fluid  (or  pus, 
if  present)  in  a  capillary  pipette.     Make  cultivations, 
tube  and  surface  plate,   and  cover-slip  preparations 
from  this  situation. 

1 8 .  Collect  a  specimen  of  the  urine  from  the  distended 
bladder  in  a  large  pipette  (in  the  manner  indicated  for 
heart  blood) ,  for  further  examination,  by  cultivations, 
microscopical    preparations,    and    chemical    analysis. 

19.  Collect  a  specimen  of  bile  from  the  gall  bladder 
in  similar  manner. 

20.  Excise  the  spleen  and  place  it  in  a  sterile  cap- 
sule.    Later,  sear  the  surface  of  this  organ;  plunge  the 
spear-headed  spatula  through  the  centre  of  the  seared 
area,  twist  is  round  between  the  finger  and  thumb,  and 
remove  it  from  the  organ.     Sufficient  material  will  be 
brought  away  in  the  eye  in  its  head  to  make  cultiva- 
tions.    A  repetition  of  the  process  will  afford  material 
for  cover-slip  preparations. 

21.  Seize  one  end  of  the  spleen  with  sterile  forceps. 
Sear  a  narrow  band  of  tissue,  right  around  the  organ 


400  POST-MORTEM    EXAMINATIONS 

and  divide  the  spleen  in  this  situation  with  a  pair  of 
scissors.  Holding  the  piece  of  spleen  in  the  forceps, 
dab  the  cut  surface  on  to  a  surface  plate  in  a  number  of 
different  spots. 

22.  In  like  manner  examine  the  other  organs — liver, 
lungs,  kidneys,  lymphatic  glands  (mesenteric,  hepatic, 
lumbar,  etc),  etc.  Prepare  cultivations  and  cover-slip 
preparations. 

23.  Dissect  out  a  long    bone  from   one  upper  and 
one  lower  limb  and  one  of  the  largest  ribs.     Prepare 
cultures  from  the  bone  marrow  in  each  case.     Set  aside 
these  bones  for  the  subsequent  preparation  of  marrow 
films. 

24.  Film  preparations  of  bone  marrow  are  best  made 
by  the  Price- Jones  method.     Seize  the  bone  in  a  pair 
of  pliers  and  squeeze  out  some  of  the  marrow ;  receive 
it  in  a  platinum  loop,  and  transfer  to  a  watch  glass  of 
dissociating  fluid  and  emulsify.     The  dissociating  fluid 
is  a  neutral  10  per  cent,  solution  of  glycerine  prepared 
as  follows : — 

Measure  out  10  c.c.  Price's  best  glycerine  and  90  c.c.  sterile 
ammonia-free  distilled  water.  Mix.  Titrate  against  ~  sodic  hy- 
drate solution  using  phenolphthalein  as  the  indicator.  The  initial 
reaction  is  usually  +  o.i  to  +  0.5;  add  the  calculated  amount  of 
—  sodic  hydrate  solution  to  neutralise. 

25.  Place  a  loopful  of  fresh  desiccating  fluid  on  a 
3X1  glass  slide ;  add  a  similar  loopful  of  the  marrow 
emulsion,  and  spread  very  gently  over  the  surface  of 
the  slip. 

26.  Allow  film  to  dry  in  the  air  (protected   from 
dust)  without  heating. 

27.  Stain  with  Jenner's  polychrome  stain  (page  97) 
for  two  and  a  half  minutes. 

28.  Wash   with   ammonia-free   distilled  water,  dry 
thoroughly  and  mount  in  xylol  balsam. 


CRANIAL  AND    SPINAL    CAVITIES  401 

Cranial  and  Spinal  Cavities. — 

29.  In  some  instances  it  may  be  necessary  (e.  g., 
experimental  inoculation  of  rabies)    to  examine  the 
cranial  cavity  or  to  remove  the  spinal  cord.     Return 
the  viscera  to  the  adbominal  cavity;  draw  the  flaps  of 
skin   together  and   secure   with   Michel's   steel   clips, 
Draw  the  copper  nails  securing  the  limbs  to  the  board, 
reverse  the  animal  and  again  nail  the  limbs  down — the 
body  now  being  dorsum  uppermost. 

30.  Make  a  longitudinal  incision  in  the  mesial  line 
from  snout  to  root  of  tail,  and  four  transverse  incisions 
— one  joining  the  roots  of  the  two  ears,  one  across  the 
body  at  the  level  of  the  spinis  of  the  scapulae,  another 
at  the  level  of  the  costal  margin  and  the  last  across  the 
upper  level  of  the  pelvis.     Reflect  these  flaps  of  skin. 

3 1 .  With  forceps  and  scalpel  dissect  out  the  muscles 
lying  in  the  furrow  on  either  side  of  the  spinal  processes. 

3  2 .  Cut  through  the  bases  of  the  transverse  processes 
with  bone  forceps.  Cut  away  the  vault  of  the  skull, 
cut  through  the  roots  of  the  nerves  and  remove  the  brain 
and  spinal  cord,  place  in  a  large  glass  dish  for  examina- 
tion. Prepare  cultivations  from  the  cerebro-spinal 
fluid.  The  removal  of  the  brain  and  cord  is  a  tedious 
process  and  during  the  dissection  it  is  difficult  to  avoid 
injury  to  these  structures. 

The  operation  is,  however,  carried  out  very 
expeditiously  and  neatly  with  the  aid  of  the  surgical 
engine  (vide  page  361).  A  small  circular  saw  is  fitted 
to  the  hand  piece.  The  bones  of  the  skull  are  cut 
through  and  the  whole  of  the  vault  removed,  exposing 
the  entire  vertex  of  the  brain.  Similarly  all  the  spinous 
processes  can  be  removed  in  one  string  by  running  the 
saw  down  first  one  side  of  the  spinal  column  and  then 
the  other.  In  this  way  ample  space  for  the  removal 
of  the  nervous  tissues  is  obtained  with  a  minimum 
of  labour. 
26 


402  POST-MORTEM   EXAMINATIONS 

33.  Having  completed  the  preparation  of  cultures 
remove  small  portions  of  various  organs  at  leisure  and 
place  each  in  separate  bottles  of  fixing  fluid  for  future 
sectioning.     Affix  to  each  bottle  a  label  bearing  all 
necessary  details  as  to  its  contents. 

34.  If  necessary,  remove  portions  of  the  organs  for 
preservation  and  display  as  museum  speciments  (vide 
page  404). 

35.  Gather  up  all  the  infected  instruments,  return 


FIG.  199. — Spear-headed  platinum  spatula  (actual  size.) 

them  to  the  steriliser,  and  disinfect  by  boiling  for  ten 
minutes. 

36.  Sprinkle  dry  saw-dust  into  the  exposed  body 
cavities  to  absorb  blood  and  fluid.     Cover  the  body 
with  blotting  or  filter  paper,  moistened  with  2  per  cent, 
lysol  solution.     Place  in  a  galvanised  iron  pail,  provided 
with  a  lid,  ready  for  transport  to  the  crematorium. 

37.  Cremate  the  cadaver  together  with  the  board 
upon  which  it  is  fixed. 

38.  Stain  the   cover-slip   preparations   by   suitable 
methods  and  examine  microscopically. 

39.  Incubate  the  cultivations  and  examine  carefully 
from  day  to  day. 

40.  Make  full  notes  of  the  condition  of  the  various 
body  cavities  and   of   the   viscera   immediately   the 
autopsy   is    completed ;  and    add    the   result   of   the 
microscopical      and      cultural     investigation     when 
available. 

As  part  of  the  card  index  system  in  use  in  the  author's 
laboratory  already  referred  to  (vide  page  335)  there  is  a 
special  yellow  card  for  P-M  notes.  On  the  face  of  the 
card  are  printed  headings  for  various  data — some  of 
which  are  sometimes  unintentionally  omitted — and 
on  the  reverse  is  a  schematic  figure  which  can  be  utilised 


AUTOPSY    RECORDS 


403 


for  indicating  the  position  of  the  chief  lesions  in  the 
cadaver  of  any  of  the  laboratory  animals. 

4 1 .  Finally,  the  results  of  the  action  of  the  organism 


<  <a  o  Q 


<  PQ   U 


or  organisms  isolated  may  be  correlated  with  the 
symptoms  observed  during  life  and  the  observations 
summarised  under  the  following  headings: 


404 

Tissue  changes: 


POST-MORTEM   EXAMINATIONS 


1.  Local — i.e.,  produced  in  the  neighbourhood  of  the  bacteria. 
Position:  (a)   At  primary  lesion. 

(b)   At  secondary  foci. 
Character:    (a)    Vascular  changes  and  tissue  ]     Acute 

reactions.  j-        or 

(b)    Degeneration  and  necrosis.        J    chronic. 

2.  General  (i.  e.,  produced  at  a  distance  from  the  bacteria,  by 

!  absorption  of  toxins) : 


FIG.  261. — Back  of  post-mortem  card. 

(a)    In  special  tissues — e.  g.,  nerve  cells  and  fibres,  secreting 

cells,  vessel  walls,  etc. 
(6)    General  effects  of  malnutrition,  etc. 
Symptoms: 

(a)   Associated  with  known  tissue  changes. 
(6)    Without  known  tissue  changes. 

Permanent  Preparations — Museum  Specimens. — 

I.  Tissues. — The  naked-eye  appearances  of  morbid 
tissues  may  be  preserved  by  the  following  method : 

i.  Remove  the  tissue  or  organ  from  the  cadaver  as 
soon  after  death  as  possible,  using  great  care  to  avoid 
distortion  or  injury. 


PERMANENT    PREPARATIONS  405 

2.  Place  it  in  a  wide-mouthed  stoppered  jar,  large 
enough  to  hold  it  conveniently,  resting  on  a  pad  of 
cotton-wool,  and  arrange  it  in  the  position  it  is  intended 
to  occupy  (but  if  it  is  intended  to  show  a  section  of  the 
tissue  or  organ,  do  not  incise  it  yet) . 

3.  Cover  with  the  Kaiserling  fixing  solution,   and 
stopper  the  jar;  allow  the  tissues  to  remain  in  this 
solution    for   from   forty-eight   hours    to    seven    days 
(according  to  size)  to  fix.    Make  any  necessary  sections. 

Kaiserling  modified  solution  is  prepared  as  follows :  . 
Weigh  out 

Potassium  acetate      .    .    .    .    .    .30  grammes. 

Potassium  nitrate 15  grammes. 

and  dissolve  in 

Distilled  water 1000  c.c. 

then  add 

Formalin 150  c.c. 

Filter. 

This  fixing  solution  can  be  used  repeatedly  so  long 
as  it  remains  clear.  Even  when  it  has  become  turbid, 
if  simple  filtration  is  sufficient  to  render  it  clear,  the 
filtrate  may  be  used  again. 

4.  Transfer  the  tissue  to  a  bath  of  methylated  spirit 
(95  per  cent.)  for  thirty  minutes  to  one  hour. 

5.  Remove  to  a  fresh  bath  of  spirit  and  watch  care- 
fully.    When  the  natural  colours  show  in  their  original 
tints,  average  time  three  to  six  hours,  remove  the  tis- 
sues from  the  spirit  bath,  dry  off  the  spirit  from  the 
cut  surfaces  by  mopping  with  a  soft  cloth,  then  transfer 
to  the  mounting  solution. 

Jore  's  mounting  solution  (modified)  consists  of 

Glycerine 500  c.c. 

Distilled  water 750  c.c. 

Formalin  ....        2  c.c. 


406  POST-MORTEM   EXAMINATIONS 

Equally  good  but  much  cheaper  is  Frost's  mounting 
solution : 

Potassium   acetate 160  grammes. 

Sodium  fluoride 80  grammes. 

Chloral  hydi  ate 80  grammes. 

Cane  sugar  (Tate's  cubes)  .    .    .    .  3,500  grammes. 

Saturated  thymol  water 8,000  c.  c. 

6.  After  twenty-four  hours  in  this  solution,  or  as 
soon  as  the  tissue  sinks,  transfer  to  a  museum  jar,  fill 
with  fresh  mounting  solution,  and  seal. 

6a.  Or  transfer  to  museum  jar  and  fill  with  liquefied 
gelatine,  to  which  has  been  added  i  per  cent,  formalin. 
Cover  the  jar  and  allow  the  gelatine  to  set.  When 
solid,  seal  the  cover  of  the  jar  in  place. 

7.  To  seal  the  museum  preparation  first  warm  the 
glass    plate    which   forms    the    cover.     This    is    most 
conveniently  done  by  placing  the  cleaned  and  polished 
cover-plate  upon  a  piece  of  asbestos  millboard  over  a 
bunsen  flame  turned  low. 

8.  Smear  an  even  layer  of  hot  cement  over  the  flange 
of  the  jar.     The  cement  is  prepared  as  follows : 

Weigh  out  and  mix  in  an  iron  ladle 

Gutta  percha  (pure) 4  parts. 

Asphaltum 5  parts. 

and  melt  together  over  a  bunsen  flame,  stirring  with  an 
iron  rod  until  solution  is  complete. 

9.  Invert  the  glass  plate  over  the  jar  and  press  down 
firmly  into  the  cement.     Place  a  piece  of  asbestos 
board  on  the  top  and  on  that  rest  a  suitable  weight 
until  the  cement  is  cold  and  has  thoroughly  set. 

10.  Trim  off  any  projecting  pieces  of  cement  with 
an  old  knife,  burr  over  the  joint  between  jar  and  cover- 
plate  with  a  hot  smooth  piece  of  metal  (e.  g.,  the  sear- 
ing iron) . 

1 1 .  Paint  a  narrow  band  of  Japan  black  to  finish  off, 
round  the  joint,  overlapping  on  to  the  cover-plate. 


PERMANENT  PREPARATIONS 


407 


II.  Tube  Cultivations  of  Bacteria. — When  showing 
typical  appearances  these  may  be  preserved,  if  not 
permanently,  at  least  for  many  years,  as  museum 
specimens,  by  the  following  method : 

1.  Take  a  large  glass  jar  25  cm.  high  by  18  cm.  diam- 
eter, with  a  firm  base  and  a  broad  flange,  carefully 
ground,  around  the  mouth.     The  jar  must  be  fitted 
with  a  disc  of  plate  glass  ground  on  one 

side,  to  serve  as  a  lid. 

2 .  Smear  a  thick  layer  of  resin  ointment 
(B.P.)   on  the  flange  around  the  mouth 
of  the  jar. 

3.  Cover  the  bottom  of  the  jar  with  a 
layer  of  cotton-wool  and  saturate  it  with 
formalin. 

4.  Remove  the  cotton- wool  plug  from 
the  culture  tubes  and  place  them,  mouth 
upward,   inside  the  jar.     (If  water  of  con- 
densation is  present  in  any  of  the  culture 
tubes,  it  should  be  removed  by  means  of 
a  capillary  pipette  before  placing  the  tubes 
in  the  formalin  chamber.) 

5.  Adjust   the   glass   disc,  ground  side 
downward,  over  the  mouth  of  the  jar  and 
secure  it  by  pressing  it  firmly  down  into 
the  ointment,  with  a  rotary  movement. 

6.  Remove  the  tubes  from  the  formalin 


.  FIG.  202. — Bui- 
chamber  after  the  lapse  of  a  week,  and    loch's  tubes. 

dry  the  exterior  of  each. 

7.  Seal  the  open  mouth  of  each  tube  in  the  blowpipe 
flame  and  label. 

If  the  cultivations  are  intended  for  museum  purposes 
when  they  are  first  planted,  it  is  more  convenient  to 
employ  Bulloch  's  tubes.  These  are  slightly  longer  than 
the  ordinary  tubes,  and  are  provided  with  a  constriction 
some  2  cm.  below  the  mouth  (Fig.  202) — a  feature  which 
renders  sealing  in  the  blowpipe  flame  an  easy  matter. 


XX.     THE  STUDY  OF  THE  PATHOGENIC 
BACTERIA. 

The  student,  who  has  conscientiously  worked  out  the 
methods,  etc.,  previously  dealt  with,  is  in  a  position  to 
make  accurate  observations  and  to  write  precise  de- 
scriptions of  the  results  of  such  observations.  He  is, 
therefore,  now  entrusted  with  pure  cultivations  of  the 
various  pathogenic  bacteria,  in  order  that  he  may 
study  the  life-history  of  each  and  record  the  results 
of  his  own  observations — to  be  subsequently  corrected 
or  amplified  by  the  demonstrator.  In  this  way  he  is 
rendered  independent  of  text-book  descriptions,  the 
statements  in  which  he  is  otherwise  too  liable  to  take 
for  granted,  without  personally  attempting  to  verify 
their  accuracy. 

During  the  course  of  this  work  attention  must  also  be 
directed,  as  occasion  arises,  to  such  other  bacteria, 
pathogenic  or  saprophytic,  as  are  allied  to  the  particular 
organisms  under  observation,  or  so  resemble  them  as  to 
become  possible  sources  of  error,  by  working  them 
through  on  parallel  lines — in  other  words  the  various 
bacteria  should  be  studied  in  "groups."  In  the  follow- 
ing pages  the  grouping  in  use  in  the  author's  elemen- 
tary classes  for  medical  and  dental  students  and  for 
candidates  for  the  Public  Health  service  is  adopted, 
since  a  fairly  long  experience  has  completely  vindicated 
the  value  and  utility  of  this  arrangement,  and  by  its 
means  a  fund  of  information  is  obtained  with  regard 
to  the  resemblances  and  differences,  morphological  and 
cultural,  of  a  large  number  of  bacteria.  The  fact  that 
some  bacteria  appear  in  more  than  one  of  these  groups, 
so  far  from  being  a  disadvantage,  is  a  positive  gain  to 
the  student,  since  with  repetition  alone  will  the  neces- 

408 


STUDY    OF   PATHOGENIC    BACTERIA  409 

sary  familiarity  with  the  cultural  characters  of  impor- 
tant bacteria  be  acquired.  The  study  of  the  various 
groups  will  of  course  vary  in  detail  with  individual 
demonstrators,  and  with  the  student's  requirements — 
the  general  line  it  should  take  is  indicated  briefly  in 
connection  with  the  first  group  only  (pages  410-411). 
This  section  should  be  carefully  worked  through  before 
the  student  proceeds  to  the  study  of  bacterioscopical 
analysis. 

It  is  customary  to  commence  the  study  of  the  patho- 
genic bacteria  with  the  Organisms  of  Suppuration. 
This  is  a  large  group,  for  all  the  pathogenic  bacteria 
possess  the  power,  under  certain  conditions,  of  initiat- 
ing purely  pyogenic  processes  in  place  of  or  in  addition 
to  their  specific  lesions,  (e.  g.,  Bacillus  tuberculosis, 
Streptococcus  lanceolatus,  Bacillus  typhosus,  etc.). 
There  are,  however,  a  certain  few  organisms  which 
commonly  express  their  pathogenicity  in  the  formation 
of  pus.  These  are  usually  grouped  together  under  the 
title  of  "pyogenic  bacteria,"  as  distinct  from  those 
which  only  occasionally  exercise  a  pyogenic  role. 

The  organisms  included  in  this  group  are : 

1.  Staphlococcus  pyogenes  albus. 

2.  Staphylococcus  pyogenes  aureus. 

3.  Staphylococcus  pyogenes  citreus. 

4.  Streptococcus  pyogenes  longus. 

5.  Micrococcus  tetragenus. 

6.  Bacillus  pyocyaneus. 

7.  Bacillus  pneumoniae. 
and  in  certain  special  tissues 

8.  Micrococcus  gonorrhoeas. 

9.  Micrococcus  intracellularis  meningitidis  (Menin- 
gococcus) . 

10.  Micrococcus  catarrhalis. 

11.  Bacillus aegypticus  (Koch- Weeks  Bacillus). 

The  group  may  with  advantage  be  subdivided  as 
indicated  in  the  following  pages : 


41 0  STUDY   OF   PATHOGENIC   BACTERIA 

I.  Pyogenic  cocci. 

Staphylococcus  pyogenes  albus. 
Staphylococcus  pyogenes  aureus. 
Staphylococcus  pyogenes  citreus. 

to  contrast  with 
Micrococcus  candicans. 
Micrococcus  agilis. 

i.  Prepare  subcultivations  from  each: 
Bouillon, 

Agar  streak, 

-D?    A  and  incubate  at  3  7°  C. 

Blood  serum, 


Litmus  milk. 
Agar  streak, 
Gelatine  stab, 
Potato. 


and  incubate  at  20°  C. 


Compare  the  naked-eye  appearances  of  the  cultures 
from  day  to  day.  Note  M.  agilis  refuses  to  grow  at 
37°C. 

2 .  Make  hanging-drop  preparations  from  the  bouillon 
and  agar  cultivations  after  twenty-four  hours'  incu- 
bation.    Examine  microscopically  and  compare.     Note 
the  locomotive  activity  of  M.  agilis  and  the  Brownian 
movement  of  the  remaining  micrococci. 

3.  Prepare  cover-slip  films  from  the  agar  cultures, 
after  twenty-four  hours'  incubation.     Stain  for  flagella 
by  the  modified  Pit  field's  method.     Note  M.  agilis  is 
the  only  micrococcus  showing  flagella. 

4.  Make  microscopical  preparations  of  each  from  all 
the  various  media  after  twenty-four  and  forty-eight 
hours    and    three    days'    incubation.     Stain   carbolic 
methylene-blue,  carbolic  fuchsin,  and  Gram's  method. 
Examine    the    films     microscopically    and    compare. 
Note  in  the  Gram   preparation,    the   Gram   negative 
character  of  certain  individual  cocci  in  each  film  pre- 
pared from  the  three  days'  growth — such  cocci  are  dead. 

5.  Stain  section  of  kidney  tissue  provided  (showing 


STUDY    OF    PATHOGENIC    BACTERIA  41! 

abscess    formation    by    Staphylococcus    aureus)     by 
Gram's  method,  and  counterstain  with  eosin. 

6.  Stain  film  preparation  of  pus  from  an  abscess 
(containing    Staphylococcus    pyogenes    aureus)    with 
carbolic  methylene-blue  and  also  by  Gram's  method, 
counterstained  with  eosin. 

7.  Inoculate1  a  white  mouse   subcutaneously  with 
three  loopfuls  of  a  forty-eight-hour  agar  cultivation  of 
the   Staphylococcus  aureus,   emulsified  with  0.2   c.c. 
sterile  broth. 

Observe  carefully  during  life,  and  when  death  occurs 
make  a  careful  post-mortem  examination. 
II.  Pyo genie  cocci. 

Micrococcus  gonorrhoeas . 

Micrococcus    intracellularis    meningitidis 

(meningococcus) . 
Micrococcus  catarrhalis. 
Micrococcus  tetragenus. 
Micrococcus  paratetragenus. 

III.  Pyo  genie  cocci. 

Streptococcus  pyogenes  longus. 
Streptococcus  of  bovine  mastitis. 
Streptococcus    lanceolatus     (Diplococcus 

pneumoniae  or  pneumococcus) . 

to  contrast  with 
Streptococcus  brevis. 
Streptococcus  lebensis. 

IV.  Pyogenic  bacilli. 

Bacillus  pneumoniae  (Friedlaender) . 
Bacillus  rhinoscleromatis. 
Bacillus  lactis  aerogenes. 

V.  Pyogenic  bacilli. 

Bacillus  pyocyaneus. 

to  contrast  with 

Bacillus  fluorescens  liquefaciens. 
Bacillus  fluorescens  non-liquefaciens. 

1  See  note  on  Vivisection  License,  page  334. 


412  STUDY    OF    PATHOGENIC    BACTERIA 

VI.  Pneumonia  group. 

Streptococcus  lanceolatus  (pneumococcus) , 
Bacillus  pneumoniae  (Friedlaender) . 
Streptococcus  pyogenes  longus. 

VII.  Diphtheroid  group. 

Bacillus  diphtherias  (Klebs-LcefHer). 
Bacillus  Hoffmanni . 
Bacillus  xerosis. 
Bacillus  septus. 

VIII.  Coli-typhoid  group. 

B.  typhi  abdominalis  (B.  typhosus). 

B.  coli  communis. 

B.  enteritidis  (Gaertner). 

to  contrast  with 
B.  aquatilis  sulcatus. 

IX.  Esckerich  group. 

B.  coli  communis  (Escherich). 
B.  coli  communior. 
B.  lactis  aerogenes. 
B.  cloacae. 

X.  Gaertner  group. 

Bacillus  enteritidis  (Gaertner). 

B.  paratyphosus  A. 

B.  paratyphosus  B. 

Bacillus  cholerae  suum  (Hog  Cholera). 

B.  psittacosis. 

XL  Eberth  group. 

B.  typhosus  (Eberth). 
B.  dysenteriae  (Shiga). 
B.  dysenteriae  (Flexner). 
B.   faecalis  alcaligines. 


STUDY   OF   PATHOGENIC    BACTERIA  413 

XII.  Spirillum  group. 

Vibrio  choleras. 
Vibrio  metschnikovi. 

to  contrast  with 

Vibrio  proteus  (Finkler  and  Prior). 
Spirillum  rubrum. 
Spirillum  rugula. 

XIII.  Anthrax  group. 

Bacillus  anthracis. 
to  contrast  with 
Bacillus  subtilis. 
Bacillus  mycoides. 
Bacillus  mesentericus  fuscus. 

XIV.  Acid  fast  group. 

Bacillus  tuberculosis  (human). 

(bovine). 

(avian). 

(fish). 

to  contrast  with 

Bacillus  phlei  (Timothy  grass  bacillus). 
Butter  bacillus  of  Rabino witch. 

XV.  Plague  group. 

Bacillus  pestis. 

B.  septicaemias  hasmorrhagicae. 

B.  suipestifer. 

XVI.  Influenza  group. 
B.  influenzas. 

Bacillus  aegypticus  (Koch- Weeks). 
Bacillus  pertussis. 

XVII.  Miscellaneous. 

Bacillus  leprae. 
Bacillus  mallei. 
Micrococcus  melitensis. 


414  STUDY   OF   PATHOGENIC    BACTERIA 

XVIII.  Streptothrix  group. 

Streptothrix  actinomycotica. 
Streptothrix  madurae. 

to  contrast  with 
Cladothrix  nivea. 

XIX.   Tetanus  group. 

Bacillus  tetani. 

Bacillus  cedematis  maligni. 

Bacillus  chauvei  (symptomatic  anthrax), 

XX.  Enter itidis  sporo genes  group. 

Bacillus  enteritidis  sporogenes. 

B.  botulinus. 

B.  butyricus.  ^ 

B.  cadaveris. 


XXI.    BACTERIOLOGICAL  ANALYSES. 

EACH  bacteriological  or  bacterioscopical  analysis  of 
air,  earth,  sewage,  various  food-stuffs,  etc.,  includes, 
as  a  general  rule,  two  distinct  investigations  yielding 
results  of  very  unequal  value : 

1.  Quantitative. 

2.  Qualitative. 

The  first  is  purely  quantitative  and  as  such  is  of 
minor  importance  as  it  aims  simply  at  enumerating 
(approximately)  the  total  number  of  bacteria  present 
in  any  given  unit  of  volume  irrespective  of  the  nature 
and  character  of  individual  organisms. 

The  second  and  more  important  is  both  qualitative 
and  quantitative  in  character  since  it  seeks  to  accurately 
identify  such  pathogenic  bacteria  as  may  be  present 
while,  incidentally,  the  methods  advocated  are  calcu- 
lated to  indicate,  with  a  fair  degree  of  accuracy,  the 
numerical  frequency  of  such  bacteria,  in  the  sample 
under  examination. 

The  general  principles  underlying  the  bacteriological 
analyses  of  water,  sewage,  air  and  dust,  soil,  milk,  ice 
cream,  meat,  and  other  tinned  stuffs,  as  exemplified 
by  the  methods  used  by  the  author,  are  indicated 
in  the  following  pages,  together  with  the  methods  of 
testing  filters  and  chemical  germicides;  and  the  tech- 
nique there  set  out  will  be  found  to  be  capable  of  expan- 
sionand  adaptation  to  any  circumstance  or  set  of  cir- 
cumstances which  may  confront  the  student. 

Controls. — The  necessity  for  the  existence  of  ade- 
quate controls  in  all  experimental  work  cannot  be 
too  urgently  insisted  upon.  Every  batch  of  plates  that 
is  poured  should  include  at  least  one  of  the  presum- 
es 


41  6  BACTERIOLOGICAL  ANALYSES 

ably  "sterile"  medium;  plate  or  tube  cultures  should  be 
made  from  the  various  diluting  fluids;  every  tube  of 
carbohydrate  medium  that  is  inoculated  should  go 
into  the  incubator  in  company  with  a  similar  but  un- 
inoculated  tube,  and  so  on. 

BACTERIOLOGICAL  EXAMINATION  OF  WATER. 

The  bacteria  present  in  the  water  may  comprise 
not  only  varieties  which  have  their  normal  habitat  in 
the  water  and  will  consequently  develop  at  20°  C.,  but 
also  if  the  water  has  been  contaminated  with  excremen- 
tal  matter,  varieties  which  have  been  derived  from,  or 
are  pathogenic  for,  the  animal  body,  and  which  will  only 
develop  well  at  a  temperature  of  37°  C.  In  order  to 
demonstrate  the  presence  of  each  of  these  classes  it  will 
be  necessary  to  incubate  the  various  cultivations  at 
each  of  these  temperatures. 

Further,  the  sample  of  water  may  contain  moulds, 
yeasts,  or  torulae,  and  the  development  of  these  will  be 
best  secured  by  plating  in  wort  gelatine  and  incubat- 
ing at  20°  C. 

1.  Quantitative.  — 

Collection  of  the  Sample.  —  The  most  suitable  vessels 
for  the  reception  of  the  water  sample  are  small  glass 
bottles,  60  c.c.  capacity,  with  narrow  necks  and  over- 
hanging glass  stoppers  (to  prevent  contamination  of 
the  bottle  necks  by  falling  dust).  These  must  be 
carefully  sterilised  in  the  hot-air  steriliser  (vide  page 


(a)  If  the  sample  is  obtained  from  a  tap  or  pipe, 
turn  on  the  water  and  allow  it  to  run  for  a  few  minutes. 
Remove  the  stopper  from  the  bottle  and  retain  it  in 
the  hand  whilst  the  water  is  allowed  to  run  into  the 
bottle  and  three  parts  fill  it.     Replace  the  stopper  and 
tie  it  down,  but  do  not  seal  it. 

(b)  If  the  sample  is  obtained  from  a  stream,  tank, 


WATER  417 

or  reservoir,  fasten  a  piece  of  stout  wire  around  the  neck 
of  the  bottle,  remove  the  stopper,  and  retain  it  in  the 
hand.  Then,  using  the  wire  as  a  handle,  plunge  the 
bottle  into  the  water,  mouth  downward,  until  it  is 
well  beneath  the  surface ;  then  reverse  it,  allow  it  to  fill, 
and  withdraw  it  from  the  water.  Pour  out  a  few  cubic 
centimetres  of  water  from  the  bottle, 
replace  the  stopper,  and  tie  it  down. 

(c)  If  the  sample  is  obtained  from 
a  lake,  river  or  the  sea;  or  when  it  is 
desired  to  compare  samples  taken  at 
varying  depths,  the  apparatus  de- 
signed by  v.  Esmarch  (Fig.  203)  is 
employed.  In  this  the  sterilised 
bottle  is  enclosed  in  a  weighted  metal 
cage  which  can  be  lowered,  by  means 
of  a  graduated  line,  until  the  required 
depth  is  reached.  At  this  point  the 
bottle  is  opened  by  a  thin  wire  cord 
attached  to  the  stopper;  when  the 
bottle  is  full  (as  judged  by  the  air 
bubbles  ceasing  to  rise)  the  pull  on  J^^oi^ng 
the  cord  is  released  and  the  tension  bottle  for  water 
of  the  spiral  spring  above  the  stopper 
again  forces  it  into  the  neck  of  the  bottle.  When  the 
apparatus  is  taken  out  of  the  water,  the  small  bottles 
are  filled  from  it,  and  packed  in  the  ice-box  mentioned 
below. 

An  inexpensive  substitute  for  Esmarch' s  bottle  can 
be  made  in  the  laboratory  thus : 

Select  a  wide-mouthed  glass  stoppered  bottle  of  about 
500  c.c.  capacity  (about  20  cm.  high  and  8  cm.  in 
diameter) . 

Remove  the  glass  stopper  and  insert  a  rubber  cork 
with  two  perforations  in  its  place. 

Through  one  perforation  pass  a  piece  of  glass  tubing 
about  5  cm.  long  and  through  the  other  a  piece  22 
27 


4i8 


BACTERIOLOGICAL  ANALYSES 


cm.  long,  reaching  to  near  the  bottom  of  the  bottle, 
each  tube  projecting  about  2.5  cm.  above  the  rubber 
stopper.     Plug  the  open  ends  of  the  tubes  with  cotton 
wool.    Secure  the  stopper  in  place  with  thin  copper  wire. 
Sterilise  the  fitted  bottle  in  the  autoclave.     Remove 
the    cotton   wool  plugs   and  connect  the  projecting 
tubes  by  a  piece  of  loosely  fitting  stout  rubber  pressure 
tubing    about    5   cm.  long,    previously 
sterilised  by  boiling. 

Take  a  piece  of  stout  rubber  cord 
about  33  cm.  long,  and  of  10  mm.  diam- 
eter (such  as  is  used  for  door  springs) 
thread  a  steel  split  ring  upon  it  and 
secure  the  free  ends  tightly  to  the  neck 
of  the  bottle  by  cord  or  catgut . 

Attach  the  cord  used  for  lowering  the 
bottle  into  the  water  to  the  split  ring 
on  the  rubber  suspender.  The  best  ma- 
terial for  this  purpose  is  cotton  insulated 
electric  wire  knotted  at  every  metre. 

Connect  the  split  ring  also  with  the 
short   piece    of   rubber  tubing  uniting 
the  two  glass  tubes  by  a  piece  of  catgut 
(or   thin    copper  wire)   of  such  length 
that  when  the  bottle  is  suspended  there 
Thresh's  '  deep    is  no   pull  upon  the  rubber  tube,   but 
bottie  sampling   which,  however,  will  be  easily  jerked  off 
when  a  sharp  pull  is  given  to  the  sus- 
pending cord. 

Now  wind  heavy  lead  tubing  about  i  cm.  diameter 
around  the  upper  part  of  the  bottle,  starting  at  the 
neck  just  above  the  shoulder.  This  ensures  the  sinking 
of  the  bottle  in  the  vertical  position  (Fig.  204). 

The  apparatus  being  arranged  is  lowered  to  the 
required  depth,  a  sharp  jerk  is  then  given  to  the  sus- 
pending cord,  which  detaches  the  rubber  tube  and  so 
opens  the  two  glass  tubes.  Water  enters  through  the 


WATER 


419 


longer  tube  and  the  air  is  expelled  through  the  shorter 
tube.  The  bubbles  of  air  can  be  seen  or  heard  rising 
through  the  water,  until  the  bottle  is  nearly  full,  a 
small  volume  of  compressed  air  remaining  in  the  neck 
of  the  bottle. 

As  the  apparatus  is  raised,  the  air  thus  imprisoned 
expands,  and  prevents  the  entry  of  more  water  from 
nearer  the  surface. 


J) 

1 

I 

FIG.  205. — Ice-box  for  transmission  of  water  samples,  etc. 

Transport  of  Sample. — If  the  examination  of  the  sam- 
ple cannot  be  commenced  immediately,  steps  must  be 
taken  to  prevent  the  multiplication  of  the  bacteria  con- 
tained in  the  water  during  the  interval  occupied  in  transit 
from  the  place  of  collection  to  the  laboratory.  To  this 
end  an  ice-box  such  as  that  shown  (in  Fig.  205)  is  essen- 
tial. It  consists  of  a  double- walled  metal  cylinder  into 
which  slides  a  cylindrical  chamber  of  sufficient  capacity 
to  accommodate  four  of  the  60  c.c.  bottles ;  this  in  turn 
is  covered  by  a  metal  disc — the  three  portions  being 


420  BACTERIOLOGICAL   ANALYSES 

bolted  together  by  thumb  screws  through  the  over- 
hanging flanges.  When  in  use,  place  the  bottles,  rolled 
in  cotton-wool,  in  the  central  chamber,  pack  the  space 
between  the  walls  with  pounded  ice,  securely  close  the 
metal  box  by  screwing  down  the  fly  nuts,  and  place  it 
in  a  felt-lined  wooden  case.  (It  has  been  shown  that 
whilst  bacteria  will  survive  exposure  to  the  temperature 
of  melting  ice,  practically  none  will  multiply  at  this 
temperature.) 

On  reaching  the  laboratory,  the  method  of  examina- 
tion consists  in  adding  measured  quantities  of  the 
water  sample  to  several  tubes  of  nutrient  media  pre- 
viously liquefied  by  heat,  pouring  plate  cultivations 
from  each  of  these  tubes,  incubating  at  a  suitable  tem- 
perature, and  finally  counting  the  colonies  which  make 
their  appearance  on  the  plates. 

Apparatus  Required: 

Plate-levelling  stand. 

Case  of  sterile  plates. 

Case  of  sterile  pipettes,  i  c.c.  (in  tenths  of  a  cubic  centimetre). 

Case  of  sterile  pipettes,  10  c.c.  (in  tenths  of  a  cubic  centimetre). 

Case  of  sterile  capsules,  25  c.c.  capacity. 

Tubes  of  nutrient  gelatine. 

Tubes  of  nutrient  agar. 

Tubes  of  wort  gelatine. 

One  250  c.c.  flask  of  sterile  distilled  water. 

Tall  cylinder  containing  2  per  cent,  lysol  solution. 

Bunsen  burner. 

Grease  pencil. 

Water-bath  regulated  at  42°  C. 

METHOD. — 

1.  Arrange    the    plate-levelling    platform    with    its 
water  compartment  filled  with  water,  at  45°  C. 

2.  Number  the  agar  tubes,  consecutively,   i  to  6; 
the  gelatine  tubes,  consecutively,  i  to  6,  and  the  wort 
tubes,  i,  2,  and  3.     Flame  the  plugs  and  see  that  they 
are  not  adherent  to  the  lips  of  the  tubes. 

3.  Place  the  agar  tubes  in  boiling .  water  until  the 


WATER  42 I 

medium  is  melted,  then  transfer  them  to  the  water- 
bath  regulated  at  42°  C.  Liquefy  the  nutrient  gelatine 
and  wort  gelatine  tubes  by  immersing  them  in  the 
same  water- bath. 

4.  Remove  the  bottle  containing  the  water  sample 
from  the  ice-box,  distribute  the  bacterial  contents 
evenly  throughout  the  water  by  shaking,  cut  the  string 
securing  the  stopper,  and  loosen  the  stopper,  but  do 
not  take  it  out. 


FIG.  206. — Withdrawing  water  from  water  sample  bottle. 

5.  Remove  one  of  the  i  c.c.  pipettes  from  the  case, 
holding  it  by  the  plain  portion  of  the  tube.     Pass  the 
graduated  portion  twice  through  the   Bunsen  flame. 
Tilt  the  bottle  containing  the  water  sample  on  the 
bench  holding  the  neck  between  the  middle  and  ring 
fingers  of  the  left  hand ;  grasp  the  head  of  the  stopper 
between  the  forefinger  and  thumb,  and  remove  it  from 
the  bottle. 

6.  Pass  the  pipette  into  the  mouth  of  the  bottle,  hold- 
ing its  point  well  below  the  surface  of  the  water  (Fig.  206) . 


422  BACTERIOLOGICAL  ANALYSES 

Suck  up  rather  more  than  i  c.c.  into  the  pipette  and 
allow  the  pipette  to  empty ;  this  moistens  the  interior  of 
the  pipette  and  renders  accurate  measurement  possible. 
Now  draw  up  exactly  i  c.c.  into  the  pipette.  Withdraw 
the  pipette  from  the  bottle,  replace  the  stopper,  and 
stand  the  bottle  upright. 

7.  Take  the  first  melted  agar  tube  in  the  left  hand, 
remove  the  cotton-wool  plug,  and  add  to  its  contents 
0.5  c.c.  of  the  water  sample  from  the  pipette;  replug 
the  tube  and  replace  it  in  the  water-bath.     In  a  similar 
manner  add  0.3  c.c.  water  to  the  contents  of  the  second 
tube,  and  0.2  c.c.  to  the  contents  of  the  third. 

8.  In  a  similar  manner  add  i  c.c.  of  the  sample  to  the 
contents  of  the  fourth  tube. 

9.  Similarly,  add  0.5  c.c.  and  o.i  c.c.  respectively  to 
the  contents  of  the  fifth  and  sixth  tubes. 

10.  Drop  the  pipette  into  the  cylinder  containing 
lysol  solution. 

11.  Mix  the  water  sample  with  the  medium  in  each 
tube  in  the  manner  described  under  plate  cultivations ; 
pour  a  plate  from  each  tube.     Label  each  plate  with 
(a)  the  distinctive  number  of  the  sample,  (b)  the  quan- 
tity of  water  sample  it  contains,  and  (c)  the  date. 

12.  Pour  the  contents  of  a  tube  of  liquefied  agar — 
not  inoculated — into  a  Petri  dish  to  act  as  a  control  to 
demonstrate  the  sterility  of  the  batch  of  agar  employed. 

13.  Allow  the  plates  to  set,  and  incubate  at  37°  C. 

14.  Empty  the  water  chamber  of  the  levelling  appa- 
ratus and  refill  it  with  ice-water. 

15.  By  means  of  the  sterile  10  c.c.  pipette  deliver 
9.9    c.c.    sterile   distilled    water   into   a   sterile   glass 
capsule. 

1 6.  Add  o.i  c.c.  of  the  water  sample  to  the  9.9  c.c. 
sterile  water  in  the  capsule.     This  will  give  a  dilution 
of  i  in  100. 

17.  Plant  the  six  tubes  of  nutrient  gelatine  in  the 
following  manner:  To  the  first  tube  add  0.5  c.c.  of  the 


WATER  423 

water  sample  direct  from  the  bottle;  to  the  second, 
0.3  c.c.;  and  to  the  third,  0.2  c.c. ;  and  pour  a  plate 
of  each  tube.  To  the  fourth  tube  add  0.5  c.c.  of  the 
diluted  water  sample  from  the  capsule;  to  the  fifth, 
0.3  c.c. ;  and  to  the  sixth,  0.2  c.c. ;  and  pour  a  plate  from 
each. 

18.  Label  each  plate  with  the  quantity  of  the  water 
sample  it  contains — that  is,  0.5  c.c.,  0.3  c.c.,  0.2  c.c., 
0.005  c-c-»  0-003  c-c->  and  0.002  c.c. 

19.  Pour  a   control    (uninoculated)    gelatine   plate. 

20.  Allow  the  plates  to  set,  and  incubate  at  20°  C. 

21.  To  the  first  tube  of  liquefied  wort  gelatine  add 
0.5  c.c.  water  sample;  to  the  second,  0.3  c.c. ;  and  to  the 
third,  0.2  c.c. 

22.  Label  the  plates,  allow  them  to  set,  and  incubate 
at  20°  C. 

23.  Count  and  record  the  number  of  colonies  that 
have  developed  upon  the  agar  at  37°  C.  after  forty- 
eight  hours'  incubation. 

24.  Note  the  number  of  colonies  present  on  each  of 
the  gelatine  and  wort  gelatine  plates  after  forty-eight 
hours'  incubation. 

25.  Replace    the  gelatine  and  wort  plates  in  the 
incubator;  observe  again  at  three  days,  four  days,  and 
five  days. 

26.  Calculate  and  record  the  number  of  organisms 
present  per  cubic  centimetre  of  the  original  water  from 
the  average  of  the  six  gelatine  plates  at  the  latest  date 
possible  up  to  seven  days — the  presence  of  liquefying 
bacteria  may  render  the  calculation  necessary  at  an 
earlier  date,  hence  the  importance  of  daily  observations. 

Method  of  Counting. — The  most  accurate  method 
of  counting  the  colonies  on  each  of  the  plates  is  by 
means  of  either  Jeffer's  or  Fakes'  counting  disc.  Each 
of  these  discs  consists  of  a  piece  of  paper,  upon  which  is 
printed  a  dead  black  disc,  subdivided  by  concentric 
circles  and  radii,  printed  in  white.  In  Jeffer's  counter 


424 


BACTERIOLOGICAL  ANALYSES 


(Fig.  207),  each  subdivision  has  an  area  of  i  square 
centimetre;  in  Fakes'  counter  (Fig.  208),  radii  divide 


FIG.  207. — Jeffer's  disc,  reduced. 


FIG.  208. — Fakes'  disc,  reduced. 

the  circle  into  sixteen  equal  sectors,  and  counting  is 
facilitated  by  concentric  circles  equidistant  from  the 
centre. 


WATER  425 

(a)  In  the  final  counting  of  each  plate,  place  the 
plate  over  the  counting  disc,  and  centre  it,  if  possible, 
making  its  periphery  coincide  with  one  or  other  of  the 
concentric  circles. 

(b)  Remove  the  cover  of  the  plate,  and  by  means  of 
a  hand  lens  count  the  colonies  appearing  in  each  of  the 
sectors  in  turn.     Make  a  note  of  the  number  present  in 
each. 

(c)  If  the  colonies  present  are  fewer  than  500,  the 
entire  plate  should  be  counted.     If,   however,   they 
exceed    this    number,    enumerate    one-half,    or    one? 
quarter  of  the  plate,  or  count  a  sector  here  and  there, 
and  from  these  figures  estimate  the  number  of  colonies 
present  on  the  entire  plate.     In  practice  it  will  be  found 
that  Fakes'  disc  is  more  suitable  for  the  former  class  of 
plate;  Jeffer's  disc  for  the  latter.     It  should  be  recol- 
lected however  that  unless  the  plates  have  been  care- 
fully levelled  and  the  medium  is  of  equal  thickness  all 
over  it  is  useless  to  try  and  average  from  small  areas — 
since  where  the  medium  is  thick  all  the  bacteria  will 
develop,  where  the  layer  is  a  thin  one,  only  a  few  bacteria 
will  find  sufficient  pabulum  for  the  production  of  visible 
colonies. 

It  will  be  noted  that  the  quantities  of  water  selected 
for  addition  to  each  set  of  tubes  of  nutrient  media 
have  been  carefully  chosen  in  order  to  yield  workable 
results  even  when  dealing  with  widely  differing  samples. 
Plates  prepared  in  agar  with  o.i  c.c.  and  in  gelatin 
with  0.02  c.c.  can  be  counted  even  when  large  numbers 
of  bacteria  are  present  in  the  sample ;  whereas  if  micro- 
organisms are  relatively  few,  agar  plate  4  and  gelatine 
plate  i  will  give  the  most  reliable  counts.  Again  the 
counts  of  the  plates  in  a  measure  control  each  other ;  for 
example,  the  second  and  third  plates  of  each  gelatine 
series  should  together  contain  as  many  colonies  as  the 
first,  and  the  second  should  contain  about  half  as 
many  more  than  the  third  and  so  on. 


426  BACTERIOLOGICAL   ANALYSES 

2.  Qualitative  Examination.— 

Collection  of  Sample. — The  water  sample  required  for 
the  routine  examination,  which  it  will  be  convenient  to 
consider  first,  amounts  to  about  no  c.c.  It  is  col- 
lected in  the  manner  previously  described  (vide  page 
416) ;  similar  bottles  are  used,  and  if  four  are  filled  the 
combined  contents,  amounting  to  about  240  c.c.,  will 
provide  ample  material  for  both  the  qualitative  and 
quantitative  examinations.  Unless  the  examination  is 
to  be  commenced  at  once,  the  ice-box  must  be  employed, 
otherwise  water  bacteria  and  other  saprophytes  will 
probably  multiply  at  the  expense  of  the  microbes  in- 
dicative of  pollution,  and  so  increase  the  difficulties 
of  the  investigation. 

In  the  routine  examination  of  water  supplies  it  is 
custdmary  to  limit  the  qualitative  examination  to  a 
search  for 

A.  B.  coli  and  its  near  allies. 

B.  Streptococci, 

organisms  which  are  frequently  spoken  of  as  microbes 
of  indication,  as  their  presence  is  held  to  be  evidence  of 
pollution  of  the  water  by  material  derived  from  the 
mammalian  alimentary  canal,  and  so  to  constitute  a 
danger  signal. 

C.  Some  observers  still  attach  importance  to  the 
presence  of  B.  enteritidis  sporogenes,  but  as  the  search 
for  this  bacterium,  (relatively  scarce  in  water)  necessi- 
tates the  collection  of  a  fairly  large  quantity  of  water  it 
is  not  usually  included  in  the  routine  examination. 

In  the  case  of  water  samples  examined  during  the 
progress  of  an  epidemic,  of  new  supplies  and  of  unknown 
waters  the  search  is  extended  to  embrace  other  mem- 
bers of  the  coli-typhoid  group;  and  on  occasion  the 
question  of  the  presence  or  absence  of  Vibriocholerae  or 
(more  rarely)  such  bacteria  as  B.  anthracis  or  B. 
tetani,  may  need  investigation. 

When  pathogenic  or  excremental  bacteria  are  pre- 


WATER  427 

sent  in  water,  their  numbers  are  relatively  few,  owing 
to  the  dilution  they  have  undergone,  and  it  is  usual  in 
commencing  the  examination,  to  adopt  one  or  other  of 
the  following  methods : 

A.  Enrichment,  in  which  the  harmless  non-pathogen- 
ic bacteria  may  be  destroyed  or  their  growth  inhibited, 
whilst  the  growth  of  the  parasitic  bacteria  is  encouraged. 

This  is  attained  by  so  arranging  the  environment, 
(i.  e.,  Media,  incubation  temperature,  and  atmosphere) 
as  to  favor  the  growth  of  the  pathogenic  organisms  at 
the  expense  of  the  harmless  saprophytes. 

B.  Concentration,  whereby  all  the  bacteria  present  in 
the  sample  of  water,  pathogenic  or  otherwise,  are  con- 
centrated in  a  small  bulk  of  fluid. 

This  is  usually  effected  by  filtration  of  the  water 
sample  through  a  porcelain  filter  candle,  and  the 
subsequent  emulsion  of  the  bacterial  residue  remaining 
on  the  walls  of  the  candle  with  a  small  measured  quan- 
tity of  sterile  bouillon. 

A.  Enrichment  Method. 

(Dealing  with  the  demonstration  of  bacteria  of  in- 
testinal origin.) 

Apparatus  Required  (Preliminary  Stage): 

Incubator  running  at  42°  C. 

Case  of  sterile  pipettes,  i  c.c.  graduated  in  tenths. 
Case  of  sterile  pipettes,  10  c.c.  graduated  in  c.c. 
Case  of  sterile  pipettes,  graduated  to  deliver  25  c.c. 
Tubes  of  bile  salt  broth  (  vide  page  180). 
Flask  of  double  strength  bile  salt  broth  (vide  page  199). 
Tubes  of  litmus  silk. 
Sterile  flasks,  250  c.c.  capacity. 
Buchner's  tubes. 
Tabloids  pyrogallic  acid. 
Tabloids  sodium  hydrate. 
Bunsen  burner. 
Grease  pencil. 
(Later  stage]: 

Incubator  running  at  37°  C. 

Surface  plates  of  nutrose  agar  (see  page  232). 


428  BACTERIOLOGICAL  ANALYSES 

Aluminum  spreader. 

Tubes  of  various  media,  including  carbohydrate  media. 

Agglutinating  sera,  etc. 

METHOD.— 

1.  Number  a  set  of  bile  salt  broth  tubes  1-5,  and 
a  duplicate  set  ia-5a. 

2.  Number  one  flask  7  and  another  8. 

3-.  To  Tubes  No.  i  and  la  add  o.i  c.c.  water  sample. 
To  Tubes  No.  2  and  2 a  add  i  c.c.  water  sample. 
To  Tubes  No.  3  and  3 a  add  2  c.c.  water  sample. 
To  Tubes  No.  4  and  4a  add  5  c.c.  water  sample. 
To  Tubes  No.  5  and  5a  add  10  c.c.  water  sample. 

4.  Put  up  all  the  tubes  in  Buchner's  tubes  and  incu- 
bate anaerobically  at  42°  C. 

NOTE. — The  bile  salt  medium  is  particularly  suitable  for  the 
cultivation  of  bacteria  of  intestinal  origin,  and  at  the  same  time 
inhibits  the  growth  of  bacteria  derived  from  other  sources. 

The  anaerobic  conditions  likewise  favor  the  multipli- 
cation of  intestinal  bacteria,  and  also -their  fermenta- 
tive activity.  The  temperature  42°  C.  destroys  or- 
dinary water  bacteria  and  inhibits  the  growth  of  many 
ordinary  mesophilic  bacteria. 

5.  Pipette  25  c.c.  of  double  strength  bile  salt  broth 
into  flask  6,  and  50  c.c.  double  strength  bile  salt  broth 
into  flask  7. 

6.  Pipette  25  c.c.  water  sample  into  flask  6,  and  50 
c.c.  water  sample  into  flask  7. 

7.  Incubate  the  two  flasks  aerobically  at  42°  C. 

8.  After  twenty-four  hours  incubation  note  in  each 
culture : 

a.  The  presence  or  absence  of  visible  growth. 

b.  The  reaction  of  the  medium  as  indicated  by  the 
colour  change,  if  any,  the  litmus  has  undergone. 

c.  The  presence   or  absence   of  gas   formation,    as 
indicated  by  a  froth  on  the  surface  of  the  medium,  and 
the  collection  of  gas  in  the  inner  "gas"  tube. 


WATER  429 

9.  Replace   those   tubes   which   show   no   signs   of 
growth    in    the    incubator.     Examine    after    another 
period  of  twenty:four  hours   (total  forty-eight  hours 
incubation)  with  reference  to  the  same  points. 

10.  Remove  culture  tubes  which  show  visible  growth 
from  the   Buchner's  tubes,  whether  acid  production 
and  gas  formation  are  present  or  not. 

11.  Examine  all  tubes  which  show  growth  by  hang- 
ing-drop preparations.     Note  such  as  show  the  presence 
of  chains  of  cocci. 

12.  Prepare  surface  plate  cultivations  upon  nutrose 
agar  from  each  tube  that  shows  growth  either  macro- 
scopically  or  microscopically,  and  incubate  for  twenty- 
four  hours  aerobically  at  3  7°  C. 

13 .  Examine  the  growth  on  the  plates  either  with  the 
naked  eye  or  with  the  help  of  a  small  hand  lens.     Prac- 
tice will  facilitate  the  recognition  of  colonies  of  the  coli 
group,  the  typhoid  group  and  the  paratyphoid  group; 
also  those  due  to  the  growth  of  streptococci.     The 
investigation    from    this    stage    proceeds    along    two 
divergent  lines  of  enquiry — the  first  being  concerned 
with  the  identity  of  the  bacilli — typhoid  bacilli,  the 
second  with  that  of  the  cocci. 

A.  B.  Coli  and  its  allies. 

14.  Pick  off  coliform  or  typhiform  colonies;  make 
streak  or  smear    subcultivations  upon  nutrient  agar; 
incubate  aerobically  for  twenty-four  hours  at  3  7°  C. 

15.  Examine  the  growth  in  each  tube  carefully  both 
macroscopically  and  microscopically.     If  the  growth  is 
impure,  replate  on  nutrose  agar,  pick  off  colonies  and 
subcultivate  again.     When  the  growth  in  a  tube  is  pure, 
add  5  c.c.  sterile  normal  saline  solution  or  sterile  broth, 
and  emulsify  the  entire  surface  growth  with  it. 

1 6.  Utilise  the  emulsion  for  the  preparation  of  a 
series  of  subcultivations  upon  the  media  enumerated 
below,  using  the  ordinary  loop  to  make  the  subcultures 
upon  solid  media,   but  adding  one-tenth  of  a  cubic 


430  BACTERIOLOGICAL   ANALYSES 

centimetre  of  the  emulsion  to  each  of  the  fluid  media 
by  means  of  a  sterile  pipette. 

Gelatine  streak. 
Agar  streak. 
Potato. 

Nutrient  broth. 
Litmus  milk. 

Dextrose  peptone  solution. 
Laevulose  peptone  solution. 
Galactose  peptone  solution. 
Maltose  peptone  solution. 
Lactose  peptone  solution. 
Saccharose  peptone  solution. 
Raffinose  peptone  solution. 
Dulcite  peptone  solution. 
Mannite  peptone  solution. 
Glycerin  peptone  solution. 
Inulin  peptone  solution. 
Dextrin  peptone  solution. 

17.  Differentiate  the  bacilli  after  isolation  by  means 
of  their  cultural  reactions  and  biological  characters  into 
members  of: 

I.  The  Escherich  Group. 

B.  coli  communis. 

B .  coli  communior. 

B.  lactis  aerogenes. 

B.  cloacae. 

II.  The  Gaertner  Group. 

Bacillus  enteritidis  (of  Gasrtner). 
B.  paratyphosus  A. 
B.  paratyphosus  B. 
Bacillus  cholerse  suum. 


WATER  431 

III.  The  Eberth  Group. 

B.  typhosus. 
B.  dysenterise  (Shiga)0 
B.  dysenteriae  (Flexner). 
B.  faecalis  alcaligines. 

18.  Confirm  these  results  by  testing  the  organisms 
isolated  against  specific  agglutinating  sera  obtained 
from  experimentally  inoculated  animals. 

If  a  positive  result  is  obtained  when  using  this 
method,  it  only  needs  a  simple  calculation  to  determine 
the  smallest  quantity  (down  to  o.i  c.c.)  of  the  sample 
that  contains  at  least  one  of  the  microbes  of  indication. 
For  instance,  if  growth  occurs  in  all  the  tubes  from  4  to 
10,  and  that  growth  is  subsequently  proved  to  be  due 
to  the  multiplication  of  B.  coli,  then  it  follows  that  at 
least  one  colon  bacillus  is  present  in  every  10  c.c.  of  the 
water  sample,  but  not  in  every  5  c.c.  If,  on  the  other 
hand,  the  presence  of  the  B.  coli  can  only  be  proved 
in  flask  No.  7,  then  the  average  number  of  colon  bacilli 
present  in  the  sample  is  at  least  one  in  every  50  c.  c. 
(i.  e.,  twenty  per  litre),  but  not  one  in  every  25  c.  c.  and 
so  on. 

The  general  outline  of  the  method  of  identifying 
the  members  of  the  coli-typhoid  group  is  given  in  the 
form  of  an  analytical  schema — whilst  the  full  differen- 
tial details  are  set  out  in  tabular  form. 


432 


BACTERIOLOGICAL  ANALYSES 


ANALYTICAL  SCHEME  FOR  ISOLATION  OF  MEMBERS  OF  THE  COLI  AND 
TYPHOID  GROUPS. 

Nutrose  agar. 


Red  colonies. 
Escherich  group. 


Blue  colonies. 
Gaertner  and  Eberth  groups. 


Lactose  peptone  solution. 


Gas. 

B.  coli  communis  and  its  allies. 

I 

Acid  and  gas  in  gluccse  peptone  solution. 

Acid  and  coagulation  in  milk. 

General  turbidity  and  indol  in  bouillon. 


No  gas. 

Gaertner  and  Eberth  groups. 
Glucose  peptone  solution. 


Gas. 

I 

Gaertner  group. 

L 


No  gas. 

I 

Eberth  group. 


Litmus  milk. 

Peptone  solution. 

1 

1 

Acid  at  first. 

General  turbidity. 

Alkaline  later. 

No  indol. 

No  coagulation. 

Serum  reaction. 

Litmus  milk.         Peptone  solution. 


Acid.  General  turbidity. 

No  coagulation.    No  indol. 

Serum  reaction. 


B.  Streptococci. 


19.  Pick  off  streptococcus  colonies  and  subcultivate 
upon  nutrient  agar  exactly  as  directed  in  steps  14,  15 
and  1 6. 

20.  Differentiate  the  streptococci  isolated  into  mem- 
bers of  the  saprophytic  group  of  short-chained  cocci, 
or  members   of  the  parasitic    (pathogenic)    group   of 
long-chained  cocci,  by  means  of  their  cultural  charac- 
ters,   and    record   their   numerical   frequency   in   the 
manner  indicated  for  the  members  of  the  coli-typhoid 
group. 


WATER 


433 


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B.  dysenteriae  (Flexn 
3.  fjeoalis  alkaligines 

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434  BACTERIOLOGICAL  ANALYSES 

21.  Determine  the  pathogenicity  for  mice  (subcuta- 
neous inoculation)  and  rabbits  (  intravenous  inocula- 
tion) of  the  streptococci  isolated. 

On  the  facing  insert  page  is  reproduced  a  blank  from 
the  author's  Laboratory  Water  Analysis  Book,  by 
means  of  which  an  exact  record  can  be  kept,  with  a 
minimum  of  labour,  of  every  sample  examined. 

B.  Concentration  Method. 

The  remaining  organisms  referred  to  on  page  426 
are  more  conveniently  sought  for  by  the  concentration 
method. 

Collection  of  the  Sample. — The  quantity  of  water 
required  for  this  method  of  examination  is  about  2  ooo 
c.c.,  and  the  vessel  usually  chosen  for  its  reception  is 
an  ordinary  blue  glass  Winchester  quart  bottle,  ster- 
ilised in  the  hot-air  oven,  and  over  this  a  paper  or  parch- 
ment cap  fastened  with  string.  The  bottle  may  be 
packed  in  a  wooden  box  or  in  an  ordinary  wicker  case. 
The  method  of  collecting  the  sample  is  identical  with 
that  described  under  the  heading  of  Quantitative 
Examination;  there  is,  however,  not  the  same  impera- 
tive necessity  to  pack  the  sample  in  ice  for  transmission 
to  the  laboratory. 

Apparatus  required: 

Sterile  Chamberland  or  Doulton  "  white  "  porcelain  open  mouth 
filter  candle,  fitted  with  rubber  washer. 

Rubber  cork  to  fit  mouth  of  the  filter  candle,  perforated  with 
one  hole. 

Kitasato  serum  flask,  2500  c.c.  capacity. 

Geryk  air  pump  or  water  force  pump. 

Wulff's  bottle,  fitted  as  wash-bottle,  and  containing  sulphuric 
acid  (to  act  as  a  safety  valve  between  filter  and  pump). 

Pressure  tubing,  clamps,  pinch-cock. 

Retort  stand,  with  ring  and  clamp. 

Rubber  cork  for  the  neck  of  Winchester  quart,  perforated  with 
two  holes  and  fitted  with  one  6  cm.  length  of  straight  glass  tubing, 
and  one  V  -shaped  piece  of  glass  tubing,  one  arm  3  2  cm.  in  length, 
the  other  52  cm.,  the  shorter  arm  being  plugged  with  cotton- wool. 
The  rubber  stopper  must  be  sterilised  by  boiling  and  the  glass 
tubing  by  hot  air,  before  use. 


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WATER 


435 


Flask  containing  250  c.c.  sterile  broth. 

Test-tube  brush  to  fit  the  lumen  of  the  candle,  enclosed  in  a 
sterile  test-tube  (and  previously  sterilised  by  dry  heat  or  by 
boiling) . 

Case  of  sterile  pipettes,  10  c.c.  in  tenths. 

Case  of  sterile  pipettes,  i  c.c.  in  tenths. 


FIG.  209. — Water  filtering  apparatus,  That  portion  of  the  figure  to  the  left 
of  the  vertical  line  is  drawn  to  a  larger  scale  than  that  on  the  right,  in  order 
to  show  details  of  SprengePs  pump. 

Case  of  sterile  pipettes,  i  c.c.  in  hundredths. 

Tubes  of  various  nutrient  media  (according  to  requirements). 

Twelve  Buchner's  tubes  with  rubber  stoppers. 

Pyrogallic  acid  tablets. 

Caustic  soda  tablets. 

METHOD.— 

i.  Fit  up  the  filtering  apparatus  as  in  the  accom- 
panying  diagram    (Fig.    209),    interposing   the   wash- 


436 


BACTERIOLOGICAL  ANALYSES 


bottle  with  sulphuric  acid  between  the  filter  flask  and 
the  force-pump  (in  the  position  occupied  in  the  diagram 
by  the  central  vertical  line) ,  and  placing  another  screw 
clamp  on  the  rubber  tubing  connecting  the  lateral  arm 
of  the  filter  flask  with  the  wash-bottle. 

2.  Filter  the  entire   2000  c.c.  of  water 
through  the  filter  candle. 

3.  When    the    filtration    is    completed, 
screw  up  the  clamps  and  so  occlude  the 
two  pieces  of  pressure  tubing. 

4.  Reverse  the  position  of  the  glass  tubes 
in   the   Wulff's    bottle    so    that   the  one 
nearest  the  air  pump  now  dips  into  the 
sulphuric  acid.  . 

5.  Slowly  open  the  metal  clamps  and 
allow  air  to  gradually  pass    through   the 
acid,  and  enter  filter  flask,  and  so  restore 
the  pressure. 

6.  Unship   the  apparatus,   remove  the 
cork  from  the  mouth  of  the  candle. 

7.  Pipette    10  c.c.  of  sterile  broth  into 
the  interior  of  the  candle,  and  by  means 
of  the  sterile  test-tube  brush   (Fig.  210) 

sterile  test-tube  emulsify  the  slimy  residue  which  lines  the 
brush.  candle,  with  the  broth. 

Practically  all  the  bacteria  contained  in  the  original 
2000  c.c.  of  water  are  now  suspended  in  10  c.c.  of 
broth,  so  that  i  c.c.  of  the  suspension  is  equivalent, 
so  far  as  the  contained  organisms  are  concerned,  to 
200  c.c.  of  the  original  water.  (Some  bacteria  will  of 
course  be  left  behind  on  the  walls  of  the  filter  and  in 
its  pores.) 

Up  to  this  point  the  method  is  identical,  irrespective 
of  the  particular  organism  whose  presence  it  is  desired 
to  demonstrate ;  but  from  this  point  onward  the  methods 
must  be  specially  adapted  to  the  isolation  of  definite 
groups  of  organisms  or  of  individual  bacteria. 


WATER  437 

The  Coli-Typhoid  Group. — 

1.  Number  nine  tubes  of  bile  salt  broth  (vide  page 
1 80),  consecutively  from  i  to  9. 

2.  To  No  i  add  i  c.c.  l  of  the  original  water  sample 

2  add  2  c.c.  \  before  the  nitration  is  com- 

3  add  5  c.c.  j  menced. 

3.  To  the  remaining  tubes  of  bile  salt  broth  add 
varying   quantities   of   the   suspension    by   means    of 
suitably  graduated  sterile  pipettes,  as  follows : 

No.  4  .  .  .0.05  c.c.  (equivalent  to    10  c.c.  of  the  original  water  sample). 

No.  5  .  .  .    o.  125  c.c.  (equivalent  to  25  c.c.  of  the  original  water  sample). 

No.  6  .  .  .0.25  c.c.  (equivalent  to    50  c.c.  of  the  original  water  sample). 

No.  7  .  .  .0.5    c.c.  (equivalent  to  100  c.c.  of  the  original  water  sample). 

No.  8  .  .  .    i  .o    c.c.  (equivalent  to  200  c.c.  of  the  original  water  sample). 

No.  9  .  .  .    2.5     c.c.  (equivalent  to  500  c.c.  of  the  original  water  sample). 

4.  Put  up  each  tube  anaerobically  in  a  Buchner's 
tube  and  incubate  at  42°  C. 

5.  The  subsequent  steps  are  identical  with  those 
described  under  the  Enrichment  method  (see  page  428 
to  431;  Steps  8  to  18). 

Alternative  Methods. — 

A  few  of  the  older  methods  for  the  isolation  of  the  members  of 
the  coli-typhoid  groups  are  refered  to  but  they  are  distinctly 
inferior  to  those  already  described. 

(A)  The  Carbolic  Method : 

1.  Take   ten   tubes   of  carbolised  bouillon  (vide  page  202)  and 
number  them  consecutively  from  i  to  10. 

2.  Inoculate   each   tube  with  a  different  amount  of  the  water 
sample  or  suspension,  as  in  the  previous  method. 

3.  Incubate  aerobically  at  37°  C. 

4.  Examine     the     culture     tubes     after     twenty-four     hours' 
incubation. 

5.  From  those  tubes  which  shows  signs  of  growth,  pour  plates 
in  the  usual  manner,  using  carbolised  gelatine  (vide  page  202)  in 
place  of  the  ordinary  gelatine,  and  incubate  at  20°  C.  for  three, 
four,  or  five  days  as  may  be  necessary. 

6.  Subucltivate  from  any  colonies  that  make  their  appearance, 
and  determine  their  identity  on  the  lines  laid  down  in  the  previous 
method. 

(B)  Parietti's  Method: 

i.  Take  nine  tubes  of  Parietti's  bouillon  (vide  page  202) — 
$.  e.,  three  each  of  those  containing  o.  i  c.c.,  0.2  c.c.,  and  0.5  c.c. 


438  BACTERIOLOGICAL   ANALYSES 

of  Parietti's  solution  respectively.     Mark  plainly  on  the  outside 
of  each  tube  the  quantity  of  Parietti's  solution  it  contains. 

2.  To  each  tube  add  a  different  amount  of  the  original  water, 
or  of  the  suspension,  and  incubate  at  37°  C. 

3 .  Examine   the   culture   tubes   after   twenty-four   and   forty- 
eight  hours'  incubation,  and  plate  in  nutrient  carbolised  or  potato 
gelatine  from  such  as  have  grown. 

4.  Pick  off  suspicious  colonies,  if  any  such  appear  on  the  plates, 
subcultivate  them  upon  the  various  media,  and  identify  them. 

(C)  Eisner's   Method:     This   method   simply   consists   in   sub- 
stituting  Eisner's  potato  gelatine    (vide  page   204)    for  ordinary 
nutrient  gelatine  in  any  of  the  previously  mentioned  methods. 

(D)  Cambier's  Candle  Method: 

Treat  a  large  volume  of  the  water  sample  by  the  concentration 
method  (vide  page  434). 

1.  Remove  the  rubber  stopper  from  the  mouth  of   the  filter 
candle,   introduce    10   c.c.   sterile  bouillon  into  its  interior,    and 
emulsify  the  bacterial  sediment;  replug  the  mouth  of  the  candle 
with  a  wad  of  sterile  cotton-wool. 

2.  Remove  the  filter  candle  from  the  filter  flask  and   insert 
it  into  the  mouth  of  a  flask  or  a  glass  cylinder  containing  sterile 
bouillon  sufficient  to  reach  nearly  up  to  the  rubber  washer  on 
the  candle. 

3.  Incubate  for  twenty-four  to  thirty-six  hours  at  37°  C. 

4.  From  the  now  turbid  bouillon  in  the  glass  cylinder  pour 
gelatine  plates  and  incubate  at  20°  C. 

5.  Subcultivate    and    identify    any    suspicious    colonies    that 
appear. 

(The  method  depends  upon  the  assumption  that  members  of 
the  typhoid  and  coli  groups  find  their  way  through  the  porcelain 
filter  from  the  interior  to  the  surrounding  bouillon  at  a  quicker 
rate  than  the  associated  bacteria.) 

B.  Enteritidis  Sporogenes.— 

1.  Transfer  5  c.c.  of  the  emulsion  from  the  filter 
candle  to  a  sterile  test-tube  and  plug  carefully. 

2.  Place  the  test-tube  in  the  interior  of  the  benzole 
bath  employed  in  separating  out  spore-bearing  organ- 
isms (vide  page  257),  and  expose  to  a  temperature  of 
80°  C.  for  twenty  minutes. 

3.  Number  ten  tubes  of  litmus  milk  consecutively 
from  i  to  10. 

4.  Remove  the  test-tube  from  the  benzole  bath  and 
shake  well  to  distribute  the  spores  evenly  through 
the  fluid. 


WATER  439 

5.  To  each  tube  of  litmus  milk  add  a  measured  quan- 
tity of  the  suspension  corresponding  to  the  amounts 
employed  in  isolating  the  coli  group  (vide  page  437). 

6.  Incubate    each    tube    anaerobically    at    37°  C. 
Anaerobic  conditions  can  be  obtained  by  putting  the 
cultures  up  in  Buchner's  tubes  or  in  Bulloch's  appa- 
ratus.    If,  however,  whole  milk  has  been  used  in  making 
the  litmus  milk  the  layer  of  cream  that  rises  to  the  sur- 
face will  be  sufficient  to  ensure  anaerobiosis ;  whilst 
if  separated  milk  has  been  employed  it  will  be  sufficient 
to  pour  a  layer  of  sterile  vaseline  or  liquid  paraffin  on  the 
surface  of  the  fluid. 

7.  Examine    after   twenty-four   hours'    incubation. 
Note  (if  B.  enteritidis  sporogenes  is  present) — 

(a)  Acid  reaction  of  the  medium  as  indicated  by 
the  colour  of  the  litmus  or  its  complete  decolourisation. 

(6)  Presence  of  clotting,  and  the  separation  of  clear 
whey. 

(c)  Presence  of  gas,  as  indicated  by  fissures  and  bub- 
bles in  the  coagulum,  and  possibly  masses  of  coagulum 
driven  up  the  tube  almost  to  the  plug. 

8.  Replace  the  tubes  which  show  no  signs  of  growth  in 
the  incubator  for  a  further  period  of  twenty-four  hours 
and  again  examine  with  reference  to  the  same  points. 

9.  Remove  those  tubes  which  give  evidence  of  growth 
from  the   Buchner's  tubes  and  carefully  pipette  off 
the  whey ;  examine  the  whey  microscopically. 

10.  Inoculate  two  guinea-pigs  each  subcutaneously 
with  0.5  c.c.  of  the  whey  and  observe  the  result. 

Vibrio  Cholerse.— 

1 .  Number  ten  tubes  of  peptone  water  consecutively 
from  i  to  10. 

2 .  To  each  of  the  tubes  of  peptone  water  add  a  meas- 
ured   quantity  of    the    suspension,    corresponding  to 
those  amounts  employed  in  isolating  the  members  of 
the  coli  group  (vide  page  43  7) . 


44-O  BACTERIOLOGICAL  ANALYSES 

3.  Incubate  aerobically  at  37°  C.  for  twenty-four 
hours.     Examine  the  tubes  carefully  for  visible  growth, 
especially  delicate  pellicle  formation,  which  if  present 
should  be  examined  microscopically  for  vibrios,  both 
by  stained  preparations  or  by  fresh  specimens  with  dark 
ground  illumination. 

4.  Inoculate  fresh  tubes  of  peptone  water  from  such  of 
the  tubes  as  exhibit  pellicle  formation — from  the  pel- 
licle itself — and  incubate  at  3  7°  C.  for  twenty-four  hours. 

5.  Test  the  peptone  water  itself  for  the  presence  of 
indol  and  nitrite  by  the  addition  of  pure  concentrated 
H2S04. 

5.  Prepare  gelatine  and  agar  plates  in  the  usual  way 
from  such  of  these  tubes  as  show  pellicle  formation. 

6.  Pick  off  from  the  plates  any  colonies  resembling 
those  of  the  Vibrio  cholerae  and  subcultivate  upon  all 
the  ordinary  laboratory  media. 

7.  Test  the  vibrio  isolated  against  the  serum  of  an  ani- 
mal immunised  to  the  Vibrio  cholerae  for  agglutination. 

B.  Anthracis. — 

1.  Transfer  5  c.c.  of  the  emulsion  from  the  filter 
candle  to  a  sterile  test-tube  and  plug  carefully. 

2.  Place  the  test-tube  in  the  interior  of  the  benzole 
bath  employed  in  separating  out  spore- bearing  organ- 
isms  (vide  page  257),  and  expose  to  a  temperature 
of  80°  C.  for  twenty  minutes. 

3.  Inoculate  a  young  white  rat  subcutaneously  (on 
the  inner  aspect  of  one  of  the  hind  legs)  with  i  c.c.  of  the 
emulsion.      Observe  during  life,   and,   if  the  animal 
succumbs,  make  a  complete  post-mortem  examination. 

4.  Melt  three  tubes  of  nutrient  agar  in  boiling  water 
and  cool  to  42°  C. 

5.  Number  the  tubes  i,  2,  and  3.     To  No.  i  add 
0.2  c.c.,  to  No.  2  add  0.3  c.c.,  and  to  No.  3  add  0.5  c.c. 
of  the  suspension,  and  pour  plates  therefrom. 

6.  Incubate  at  37°  C.  for  twenty-four  or  forty -eight 
hours. 


MILK  441 

7.  Pick  off  any  colonies  resembling  those  of  anthrax 
and  subcultivate  on  all  the  ordinary  laboratory  media. 

8.  Inoculate  another  young  white  rat  as  in  3,  using 
two  loopfuls  of  the  agar  subcultivation  emulsified  with 
i  c.c.  sterile  bouillon.     Observe  during  life,  and  if  the 
animal    succumbs,    make    a    complete    post-mortem 
examination. 

B.  Tetani.— 

1.  Proceed  as  detailed  above  in  steps  i  and  2  for 
the  isolation  of  the  B.  anthracis. 

2.  Add  i  c.c.  of  the  suspension  to  each  of  three  tubes 
of  glucose  formate  broth,  and  incubate  anaerobically 
in  Buchner's  tubes  at  37°  C. 

3.  From  such  of  the  tubes  as  show  visible  growth 
(with  or  without  the  production  of  gas)  after  twenty- 
four  hours'  incubation  inoculate  guinea-pigs,   subcu- 
taneously  (under  the  skin  of  the  abdomen),  using  o.i 
c.c.  of  the  bouillon  cultivation  as  a  dose.     Observe 
carefully  during  life,   and,   if  death  occurs,   make  a 
complete  post-mortem  examination. 

4.  From  the  same  tubes  pour  agar  plates  and  in- 
cubate anaerobically  in  Bulloch's  apparatus,  at  37°  C. 

5.  Subcultivate  suspicious  colonies  on  the  various 
media,  incubate  anaerobically,  making  control  cultiva- 
tions on  glucose  formate  agar,   stab  and  streak,   to 
incubate  aerobically  and  carry  out  further  inoculation 
experiments  with  the  resulting  growths. 

EXAMINATION  OF  MILK. 

"One-cow"  or  "whole"  milk,  if  taken  from  the 
apparently  healthy  animal  (that  is,  an  animal  without 
any  obvious  lesion  of  the  udder  or  teats)  with  ordinary 
precautions  as  to  cleanliness,  avoidance  of  dust,  etc., 
contains  but  few  organisms.  In  dealing  with  one-cow 
milk,  from  a  suspected,  or  an  obviously  diseased  animal, 
a  complete  analysis  should  include  the  examination 
(both  qualitative  and  quantitative)  of  samples  of 


442  BACTERIOLOGICAL  ANALYSES 

(a)  fore-milk,  (b)  mid-milk,  (c)  strippings,  and,  if  possi- 
ble, from  each  quarter  of  the  udder.  "  Mixed  "  milk,  on 
the  other  hand,  by  the  time  it  leaves  the  retailer's 
hands,  usually  contains  as  many  micro-organisms  as 
an  equal  volume  of  sewage  and  indeed  during  the  ex- 
amination it  is  treated  as  such. 

It  is  possible  however  to  collect  and  store  mixed 
milk  in  so  cleanly  a  manner  that  its  germ  content  does 
not  exceed  5000  microorganisms  per  cubic  centimetre. 
Such  comparative  freedom  from  extraneous  bacteria  is 
usually  secured  by  the  purveyor  only  when  he  resorts 
to  the  process  of  pasteurisation  (heating  the  milk  to 
65°  C.  for  twenty  minutes  or  to  77°  C.  for  one  minute) 
or  the  simpler  plan  of  adding  preservatives  to  the 
milk.  Information  regarding  the  employment  of  these 
methods  for  the  destruction  of  bacteria  should  always 
be  sought  in  the  case  of  mixed  milk  samples,  and  in 
this  connection  the  following  tests  will  be  found  useful : 

1.  Raw  Milk  (Saul). 

To  10  c.c.  milk  in  a  test  tube,  add  i  c.c.  of  a  i  per  cent,  aqueous 
solution  of  ortol  (ortho-methyl-ammo-phenol  sulphate),  recently 
prepaied  and  mix.  Next  add  0.2  c.c.  of  a  3  per  cent,  peroxide  of 
hydrogen  solution.  The  appearance  of  a  brick  red  color  within 
30  seconds  indicates  raw  milk.  Milk  heated  to  74°  C.  for  thirty 
minutes  undergoes  no  alteration  in  color;  if  heated  to  75°  C.  for  ten 
minutes  only,  the  brick  red  color  appears  after  standing  for  about 
two  minutes. 

2.  Boric  Acid. 

Evaporate  to  dryness,  50  c.c.  of  the  milk  which  has  been 
rendered  slightly  alkaline  to  litmus,  then  incinerate. 

Dissolve  in  distilled  water,  add  slight  excess  of  dilute  hydrochloric 
acid  and  again  evaporate  to  dryness. 

Dissolve  the  residue  in  a  small  quantity  of  hot  water  and  moisten 
a  piece  of  turmeric  paper  with  the  solution.  Dry  the  turmeric 
paper.  Rose  or  cherry-red  color  =  borax  or  boric  acid. 

3.  Formaldehyde  (Hehner). 

To  10  c.c.  milk  in  a  test  tube  add  5  c.c.  concentrated  commercial 
sulphuric  acid  slowly,  so  that  the  two  fluids  do  not  mix.  Hold  the 
tube  vertically  and  agitate  very  gently.  Violet  zone  at  the  junc- 
tion of  the  two  liquids  =  formaldehyde. 

4.  Hydrogen  Peroxide. 

To  10  c.c.  milk  (diluted  with  equal  quantities  of  water)  in  a  test 


MILK 


443 


tube  add  0.4  c.c.  of  a  4  per  cent,  alcoholic  solution  of  benzidine  and 
0.2  c.c.  acetic  acid.  Blue  coloration  of  the  mixture  =  hydrogen 
peroxide. 

5.  Salicylic  Acid. 

Precipitate  the  caseinogen  by  the  addition  of  acetic  acid  and 
filter.  To  the  filtrate  add  a  few  drops  of  i  per  cent,  aqueous  solu- 
tion of  ferric  chloride.  Purple  coloration  =  salicylic  acid. 

6.  Sodium  Carbonate  or  Bicarbonate. 

To  10  c.c.  of  the  milk  in  a  test  tube  add  roc.c.  of  alcohol  and 
0.3  c.c.  of  a  i  per  cent,  alcoholic  solution  of  rosolic  acid.  Brownish 
color  =  pure  milk ;  rose  color  =  preserved  milk. 

Quantitative.— 

Collection  of  Sample. — 

The  apparatus  used  for  the  collection  of  a  retail 
mixed  milk  sample  consists  of  a  cylindrical  copper  case, 
1 6  cm.  high  and  9  cm.  in  diameter,  provided  with  a 


E 


\"""  -; 


FIG.  211. — Milk-collecting  bottle  and  dipper  in  case. 

"pull-off"  lid,  containing  a  milk  dipper,  also  made  of 
copper;  and  inside  this,  again,  a  wide-mouthed,  stop- 
pered glass  bottle  of  about  250  c.c.  capacity  (about  14 
cm.  high  by  7  cm.  diameter),  having  a  tablet  for  notes, 
sand-blasted  on  the  side.  The  copper  cylinder  and  its 


444  BACTERIOLOGICAL  ANALYSES 

contents,  secured  from  shaking  by  packing  with  cotton- 
wool, are  sterilised  in  the  hot-air  oven  (Fig.  26). 
When  collecting  a  sample, 

1.  Remove  the  cap  from  the  cylinder. 

2.  Draw  out  the  cotton- wool. 

3.  Lift  out  the  bottle  and  dipper  together. 

4.  Receive  the  milk  in  the  sterile  dipper,  and  pour  it 
directly  into  the  sterile  bottle. 

5.  Enter  the  particulars  necessary  for  the  identi- 
fication of  the  specimen,  on  the  tablet,  with  a  lead 
pencil,  or  pen  and  ink. 

6.  Pack  the  apparatus  in  the  ice-box  for  transmission 
to  the  laboratory  in  precisely  the  same  manner  as  an 
ordinary  water  sample. 

"Whole"  milk  may  with  advantage  be  collected  in 
the  sterile  bottle  directly  since  the  mouth  is  sufficiently 
wide  for  the  milker  to  direct  the  stream  of  milk  into  it. 

Condensed  milk  must  be  diluted  with  sterile  distilled 
water  in  accordance  with  the  directions  printed  upon 
the  label,  then  treated  as  ordinary  milk. 

Apparatus  Required: 

Case  of  sterile  capsules  (25  c.c.  capacity). 

Case  of  sterile  graduated  pipettes,  10  c.c.  (in  tenths  of  a  cubic 

centimetre) . 
Case  of  sterile  graduated  pipettes,    i   c.c.  (in  tenths  of  a  cubic 

centimetre) . 

Flask  containing  250  c.c.  sterile  bouillon. 
Tall  cylinder  containing  2  per  cent,  lysol  solution. 
Plate-levelling  stand. 
Case  of  sterile  plates. 

Tubes  nutrient  gelatine  or  gelatine  agar. 
Tubes  of  wort  gelatine. 
Tubes  of  nutrient  agar. 
Water-bath  regulated  at  42°  C. 
Bunsen  burner. 
Grease  pencil. 

METHOD.— 

i.  Arrange  four  sterile  capsules  in  a  row;  number 
them  I,  II,  III,  and  IV. 


MILK  445 

2.  Fill  9  c.c.  sterile  bouillon  into  the  first,  and  9.9 
c.c.  bouillon  into  each  of  the  three  remaining  capsules. 

3.  Remove  i  c.c.  milk  from  one  of  the  bottles  by 
means  of  a  sterile  pipette  and  add  it  to  the  bouillon  in 
capsule  I;  mix  thoroughly  by  repeatedly  filling  and 
emptying  the  pipette. 

4.  Remove  o.i  c.c.  of  the  milky  bouillon  from  cap- 
sule I,  add  it  to  the  contents  of  capsule  II,  and  mix  as 
before. 

5.  In  like  manner  add  o.i  c.c.  of  the  contents  of 
capsule  II  to  capsule  III;  and  then  o.i  c.c.  of  the  con- 
tents of  capsule  III  to  capsule  IV. 

Then  i  c.c.  of  dilution      I  contains  o.i  c.c.  milk  sample, 

i  c.c.  of  dilution    II  contains  o.ooi  c.c.  milk  sample, 

i  c.c.  of  dilution  III  contains  o.ooooi      c.c.  milk  sample, 
i  c.c.  of  dilution  IV  contains  o.ooooooi  c.c.  milk  sample. 

6.  Melt  the  gelatine  and  the  agar  tubes  in  boiling 
water;  then  transfer  to  the  water-bath  and  cool  them 
down  to  42°  C. 

7.  Number  the  gelatine  tubes  consecutively  i  to  12. 

8.  Inoculate  the  tubes  with  varying  quantities  of  the 
material  as  follows: 

To  tube  No.     i  add  i.o  c.c.  of  the  milk  sample. 

2  add  o.  i  c.c.  of  the  milk  sample. 

3  add  i.o  c.c.  from  capsule  I. 

4  add  o.  i  c.c.  from  capsule  I. 

5  add  i.o  c.c.  from  capsule  II 

6  add  o.  i  c.c.  from  capsule  II. 

7  add  0.5  c.c.  from  capsule  III. 

8  add  0.3  c.c.  from  capsule  III. 

9  add  0.2  c.c.  from  capsule  III. 

10  add  0.5  c.c.  from  capsule  IV. 

11  add  0.3  c.c.  from  capsule  IV. 

12  add  0.2  c.c.  from  capsule  IV. 

9.  Pour  plates  from  the  gelatine  tubes;  label,  and 
incubate  at  20°  C. 

10.  Liquefy  five  wort  gelatine  tubes  and  to  them 
add  i.o  c.c.  of  the  milk  sample  and  a  similar  quantity 
of  the  diluted  milk  from  capsules  I,  II,  and  III  and  IV 
respectively. 


446  BACTERIOLOGICAL  ANALYSES 

11.  Pour  plates  from  the  wort  gelatine;  label,  and 
incubate  at  20°  C. 

12.  Inoculate  the  liquefied  agar  tubes  as  follows: 

To  tube  No.     i  add  o .  i  c.c.  of  the  milk  sample. 

2  add  o.  i  c.c.  from  capsule  I. 

3  add  o.  i  c.c.  from  capsule  II. 

4  add  o.  i  c.c.  from  capsule  III. 

5  add  i.o  c.c.  from  capsule  IV.    ^ 

6  add  o.  i  c.c.  from  capsule  IV.    J 

13.  Pour  plates  from  the  agar  tubes;  label,  and  in- 
cubate at  37°  C. 

14.  After  twenty-four  hours'  incubation  "inspect," 
and  after  forty-eight  hours'  incubation,  "count"  the 
agar  plates  and  estimate  the  number  of  "organisms 
growing  at  37°  C."  present  per  cubic  centimetre  of  the 
sample  of  milk. 

15.  After    three,    four,    or    five    days'    incubation, 
"count"  the. gelatine  plates  and  estimate  therefrom 
the  number  of  "organisms  growing  at  20°  C."  present 
per  cubic  centimetre  of  the  sample  of  milk. 

1 6.  After    a    similar    interval    "count"    the    wort 
gelatine  plates  and  estimate  the  number  of  moulds 
and  yeasts  present  per  cubic  centimetre  of  the  sample 
of  milk. 

NOTE. — Many  observers  prefer  to  employ  gelatine  agar  (see  page 
193)  for  the  quantitative  examination.  In  this  case  gelatine- 
agar  plates  should  be  poured  from  tubes  containing  the  quantities 
of  material  indicated  in  step  8,  incubated  at  28°  C.  to  30°  C. 
and  after  five  days  the  "total  number  of  organisms  developing  at 
28°  C."  recorded. 

Qualitative. — The  qualitative  bacteriological  exam- 
ination of  milk  is  chiefly  directed  to  the  detection  of  the 
presence  of  one  or  more  of  the  following  pathogenic 
bacteria  and  when  present  to  the  estimation  of  their 
numerical  frequency. 

Members  of  the  Coli-typhoid  group. 

Vibrio  cholerae. 

Streptococcus  pyogenes  longus. 

Micrococcus  melitensis. 


MILK  447 

Staphylococcus  pyogenes  aureus. 

Bacillus  enteritidis  sporogenes. 

Bacillus  diphtheriae. 

Bacillus  tuberculosis. 

Some  of  these  occur  as  accidental  contaminations, 
either  from  the  water  supply  to  the  cow  farm,  or  from 
the  farm  employees,  whilst  others  are  derived  directly 
from  the  cow. 

In  milk,  as  in  water  examinations,  two  methods  are 
available,  viz. :  Enrichment  and  Concentration — the 
former  is  used  for  the  demonstration  of  bacteria  of  in- 
testinal origin,  the  latter  for  the  isolation  of  the  micro- 
organisms of  diphtheria  and  tubercle.  The  first  essen- 
tial in  the  latter  process  is  the  concentration  of  the 
bacterial  contents  of  a  large  volume  of  the  sample  into 
a  small  compass;  but  in  the  case  of  milk,  thorough 
centrifugalisation  is  substituted  for  filtration. 

Apparatus  Required: 

A  large  centrifugal  machine.  This  machine,  to  be  of  real 
service  in  the  bacteriological  examination  of  milk,  must  conform 
to  the  following  requirements: 

1.  The  centrifugal  machine  must  be  of  such  size,  and  should 

carry  tubes  or  bottles  of  such  capacity,  as  to  enable 
from  200  to  500  c.c.  of  milk  to  be  manipulated  at  one 
time. 

2.  The  rate  of  centrifugalisation  should  be  from  2500  to 

3000  revolutions  per  minute. 

3.  The  portion  of  the  machine  destined  to  carry  the  tubes 

should  be  a  metal  disc,  of  sufficient  weight  to  ensure 
good  "flank"  movement,  continuing  over  a  con- 
siderable period  of  time.  In  other  words,  the  machine 
should  run  down  very  gradually  and  slowly  after  the 
motive  power  is  removed,  thus  obviating  any  dis- 
turbance of  the  relative  positions  of  particulate 
matter  in  the  solution  that  is  being  centrifugalised. 

4.  The  machine  should  preferably  be  driven  by  electricity, 

or  by  power,  but  in  the  case  of  hand-driven  machines — 
(a)  The  gearing  should  be  so  arranged  that  the 
requisite  speed  is  obtained  by  not  more  than 
forty  or  fifty  revolutions  of  the  crank  handle 
per  minute,  so  -that  it  may  be  maintained 
for  periods  of  twenty  or  thirty  minutes 
without  undue  exertion. 


448 


BACTERIOLOGICAL  ANALYSES 

(b)   The  handle  employed  should  be  provided  with 
a  special  fastening  (e.  g.,  a  clutch  similar  to 
that    employed     for    the    free    wheel    of    a 
bicycle),  or  should  be  readily  detachable  so 
that,  on  ceasing  to  turn,  the  handle  should 
not,  by  its  weight  and  air  resistance,  act  as  a 
brake  and  stop  the  machine  too  suddenly. 
One  of  the  few  satisfactory  machines  of  this  class  is  shown 
in  figure  212. 


^~gJ^S^S=£:^^r:=^g^^^-^=^^^^::z::==^'=^    ^=j 

FIG.  212. — Electrically    driven    centrifugal   machine,  with   flexible 
(broken)  spindle  encircled  by  the  field  magnets  of  the  motor. 

Sterile  centrifugal  tubes,  of  some  60-70  c.c.  capacity,  tapering 
to  a  point  at  the  closed  end,  plugged  with  cotton-wool. 

Small  centrifugal  machine  to  run  two  tubes  of  10  c.c.  capacity 
at  2500  to  3000  revolutions  per  minute  preferably  driven  by 
electricity,  of  the  type  figured  on  page  327  (Fig.  162). 

Sterile  centrifugal  tubes  of  10  c.c.  capacity  with  the  distal 
extremity  contracted  to  a  narrow  tube  and  graduated  in  hundredths 
of  a  cubic  centimetre  (Fig.  213). 

Sterilised  cork  borer. 

Case  of  sterile  pipettes,  10  c.c.  (in  tenths  of  a  cubic  centimetre). 


MILK 


449 


Case  of  sterile  pipettes,  i  c.c.  (in  tenths  of  a  cubic  centimetre). 

Sterile  teat  pipettes. 

Flask  of  sterile  normal  saline  solution. 

METHOD.— 

i.  Fill  50  c.c.  of  the  milk  sample  into  each  of  four 
tubes,  and  replace  the  cotton- wool  plugs  by  solid  rubber 
stoppers  (sterilised  by  boiling) ,  and  fit  the  tubes  in  the 
centrifugal  machine. 

NOTE. — One  or  two  cubic  centimetres  of  paraffinum  liquidum  in- 
troduced into  the  buckets  of  the  centrifuge  before  the  glass  tubes 
are  inserted  will  obviate  any  risk  of  breakage  to  the  latter. 


FIG.  213. — Milk  sediment- 
ing  tubes. 


FIG.  2 14. — Milk  in  centrifuge 
tube. 


2.  Centrifugalise  the  milk  sample  for  thirty  minutes 
at  a  speed  of  2500  revolutions  per  minute. 

3 .  Remove  the  motive  power  and  allow  the  machine 
to  slow  down  gradually. 

4.  Remove  the  tubes  of  milk  from  the  centrifuge. 
Each  tube  will  now  show  (Fig.  214) : 

(a)  A  superficial  layer  of  cream  (varying  in  thickness 
with   different   samples)   condensed  into  a  semi-solid 
29 


450  BACTERIOLOGICAL  ANALYSES 

mass,  which  can  be  shown  to  contain  some  organisms 
and  a  few  leucocytes. 

(b)  A  central  layer  of  separated  milk,  thin,  watery, 
and  opalescent,  and  containing  extremely  few  bacteria, 

(c)  A  sediment  or  deposit  consisting  of  the  great 
majority  of  the   contained   bacteria  and   leucocytes, 
together  with  adventitious  matter,  such  as  dirt,  hair, 
epithelial  cells,  faecal  debris,  etc, 

5.  Withdraw   the   rubber   stopper   and    remove    a 
central  plug  of  cream  from  each  tube  by  means  of  a 
sterile  cork  borer;  place  these  masses  of  cream  in  two 
sterile  capstiles.     Label  C1  and  C2. 

6.  Remove  all  but  the  last  one  or  two  c.c.  of  separated 
milk  from  each  tube,  by  means  of  sterile  pipettes. 

7.  Mix  the  deposits  thoroughly  with  the  residual 
milk,  pipette  the  mixture  from  each  pair  of  tubes  into 
one  sterile  10  c.c.  tube  (graduated)  by  means  of  sterile 
teat  pipettes,  then  fill  to  the  10  c.c.  mark  with  sterile 
normal  saline  solution  and  mix  together.     Label  D1  and 
D3. 

8.  Place  the  two  tubes  of  mixed   deposit  in  the 
centrifuge,  adjust  by  the  addition  or  subtraction  of 
saline  solution  so  that  they  counterpoise  exactly,  and 
centrifugalise  for  ten  minutes. 

NOTE. — Each  tube  now  contains  the  deposit  from  100  c.c.  of 
the  milk  sample  and  the  amount  can  be  read  off  in  hundredths 
of  a  centimetre.  The  multiplication  of  this  figure  by  100  will 
give  the  amount  of  "Apparent  Filth,"  in  "parts  per  million" — the 
usual  method  of  recording  this  quality  of  milk. 

9.  Pipette  off  all  the  supernatant  fluid  and  invert  the 
tube  to  drain  on  to  a  pad  of  sterilised  cotton-wool, 
contained  in  a  beaker.     (This  wool  is  subsequently 
cremated.) 

10.  Examine    both   cream    (C1)    and   deposit    (D1) 
microscopically — 

(a)  In  hanging-drop  preparations. 

(b)  In  film  preparations  stained  carbolic  methylene- 


MILK  451 

blue,  by  Gram 's  method,  by  Neisser  's  method,  and  by 
Ziehl-Neelsen  's  method. 

Note  the  presence  or  absence  of  altered  and  unaltered 
vegetable  fibres ;  pus  cells,  blood  discs ;  cocci  in  groups 
or  chains,  diphtheroid  bacilli,  Gram  negative  bacilli  or 
cocci,  spores  and  acid  fast  bacteria. 

ii.  Adapt  the  final  stages  of  the  investigation  to 
the  special  requirements  of  each  individual  sample, 
thus: 

1.  Members  of  the  Coli=typhoid  Group. — 

1.  Emulsify    the  deposit  from  the  second  centrifu- 
gal tube  (D2)  with  10  c.c.  sterile  bouillon  and  inocu- 
late three  tubes  of  bile  salt  broth  as  follows: 

To  Tube  No.  i  add  2  .  5  c.c.  milk  deposit  emulsion  (=25  c.c.  orig- 
inal milk.) 

To  Tube  No.  2  add  i.o  c.c.  milk  deposit  emulsion  (  =  10  c.c.  orig- 
inal milk.) 

To  Tube  No.  3  add  o .  5  c.c.  milk  deposit  emulsion  ( =  5  c.c.  orig- 
inal milk.) 

2.  Inoculate  tube  of  bile  salt  broth  No.  4  with  i  c.c. 
of  the  original  milk. 

3.  Inoculate  further  tubes  of  bile  salt  broth  with 
previously  prepared  dilutions  (see  page  445)  as  follows : 

To  tube  No.     5  add  i.o  c.c.  from  capsule  I. 
To  tube  No.     6  add  o.  i  c.c.  from  capsule  I. 
To  tube  No.     7  add  i.o  c.c.  from  capsule  II. 
To  tube  No.     8  add  o.  i  c.c.  from  capsule  II. 
To  tube  No.     9  add  i.o  c.c.  from  capsule  III. 
To  tube  No.  10  add  o.  i  c.c.  from  capsule  III. 
To  tube  No.  n  add  i.o  c.c.  from  capsule  IV. 
To  tube  No.  12  add  o.  i  c.c.  from  capsule  IV. 

and  incubate  anaerobically  (in  Buchner's  tubes)  at 
42°  C.  for  a  maximum  period  of  forty-eight  hours. 

4.  If  growth  occurs  complete  the  investigation  as 
detailed  under  the  corresponding  section  of  water  ex- 
amination (seepages  428  to  431). 

NOTE. — The  B.  coli  communis,  derived  from  the  alvine  dis- 
charges of  the  cow,  is  almost  universally  present  in  large  or  small 


452  BACTERIOLOGICAL   ANALYSES 

numbers,  in  retail  milk.  Its  detection,  therefore,  unless  in  enor- 
mous numbers,  (when  it  indicates  want  of  cleanliness) ,  is  of  little 
value. 

2.  Vibrio    Cholerse. — Inoculate    tubes    of    peptone 
water  by  using  the  same  amounts  as  in  the  search 
for  members  of  the  Coli-typhoid  groups  (vide  ante  1-3) ; 
incubate  aerobically  at  37°  C.  and  complete  the  exam- 
ination as  detailed  under  the  corresponding  section  of 
water  examination  (see  page  43  9) . 

3.  B.    Enteritidis    Sporogenes. — Inoculate   tubes    of 
litmus  milk  with  similar  amounts  to  those  used  in  the 
previous  searches,  omitting  tube  No.  i  (vide  ante  1-3) 
place  in  the  differential  steriliser  at  80°  C.  for  ten 
minutes  and  then  incubate  anaerobically  at  37°  C.  for 
a  maximum  period  of  forty -eight  hours.     Complete 
the  investigation  as  detailed  under  the  corresponding 
section  of  water  examination  (see  page  438). 

4.  B.  Diphtheria^.— 

(A)  i.  Plant  three  sets  of  serial  cultivations,  twelve 
tubes  in  e'ach  set,  from  (a)  cream  C2,  (b)  deposit  D1 
upon  oblique  inspissated  blood-serum,  and  incubate 
at370C. 

2.  Pick  off  any  suspicious  colonies  which  may  have 
made  their  appearance  twelve  hours  after  incubation, 
examine  microscopically  and  subcultivate  upon  blood- 
serum  and  place  in  the  incubator;  return  the  original 
tubes  to  the  incubator. 

3.  Repeat  this  after  eighteen  hours'  incubation. 

4.  From    the    resulting    growths    make    cover-slip 
preparations  and  stain  carbolic  methylene-blue,  Neis- 
ser's  method,  Gram's  method.     Subcultivate  such  as 
appear  to  be  composed  of  diphtheria  bacilli  in  glucose 
peptone  solution.     Note  those  in  which  acid  produc- 
tion takes  place. 

5.  Inoculate  guinea-pigs   subcutaneously  with   one 
or  two  cubic  centimetres  forty-eight-hour-old  glucose 


MILK  453 

bouillon  cultivation  derived  from  the  first  subcultiva- 
tion  of  each  glucose  fermenter,  and  observe  the  result. 

6.  If  death,  apparently  from  diphtheritic  toxaemia, 
ensues,  inoculate  two  more  guinea  pigs  with  a  similar 
quantity  of  the  lethal  culture.     Reserve  one  animal 
as  a  control  and  into  the  other  inject  1000  units  of 
antidiphtheritic  serum.     If  the  control  dies  and  the 
treated  animal  survives,  the  proof  of  the  identity  of 
the  organism  isolated  with  the  Klebs-LcefBer  bacillus 
becomes  absolute. 

7.  Inoculate  guinea-pigs  subcutaneously  with  filtered 
glucose  bouillon  cultivations   (toxins    ?)   and  observe 
the  result. 

(B)    i.  Emulsify  the  remainder  of  the  deposit  with 

5  c.c.  sterile  bouillon  and  inoculate  two  guinea-pigs, 
thus:  guinea-pig  a,  subcutaneously  with  i  c.c.  emul- 
sion; guinea-pig  b,  subcutaneously  with  2  c.c.  emulsion; 
and  observe  the  result. 

2.  If  either  or  both  of  the  inoculated  animals  suc- 
cumb, make  complete  post-mortem  examination  and 
endeavour  to  isolate  the  pathogenic  organisms  from 
the  local  lesion.  Confirm  their  identity  as  in  A  5  and 

6  (vide  supra). 

5.  Bacillus  Tuberculosis.— 

(A)  i.  Inoculate  each  of  three  guinea-pigs  (previ- 
ously tested  with  tuberculin,  to  prove  their  freedom 
from  spontaneous  tuberculosis)  subcutaneously  at  the 
inner  aspect  of  the  bend  of  the  left  knee,  with  i  c.c. 
of  the  deposit  emulsion  remaining  in  one  or  other 
tube  D^r  D2). 

2.  Introduce  a  small  quantity  of  the  cream  into  a 
subcutaneous  pocket  prepared  at  the  inner  aspect  of 
the  bend  of  the  right  knee  of  each  of  these  three  ani- 
mals.    Place  a  sealed  dressing  on  the  wound. 

3.  Observe  carefully,  and  weigh  accurately  each  day. 

4.  Kill  one  guinea-pig  at  the  end  of  the  second 


454 


BACTERIOLOGICAL  ANALYSES 


week  and  make  a  complete  post-mortem  examination. 
5.  If  the  result  of  the  examination  is  negative  or 


FIG.  215. — Cadaver  of  guinea-pig  experimentally  infected  with 
B.  tuberculosis. 

inconclusive,  kill  a  second  guinea-pig  at  the  end  of  the 
third  week  and  examine  carefully. 

6.  If  still  negative  or  inconclusive,   kill  the  third 
guinea-pig  at  the  end  of  the  sixth  week.     Make  a  care- 


MILK  455 

ful  post-mortem  examination.  Examine  material  from 
any  caseous  glands  microscopically  and  inoculate 
freely  on  to  Dorset's  egg  medium. 

NOTE. — Every  post-mortem  examination  of  animals  infected 
with  tuberculous  material  should  include  the  naked  eye  and 
microscopical  examination  of  the  popliteal,  superficial  and  deep 
inguinal,  iliac,  lumbar  and  axillary  glands  on  each  side  of  the 
body,  also  the  retrohepatic,  bronchial  and  sternal  glands,  the 
spleen,  liver  and  lungs  (Fig.  215). 

(B)  i.  Intimately  mix  all  the  available  cream  and 
deposit  from  the  milk  sample,  and  transfer  to  a  sterile 
Erlenmeyer  flask. 

2.  Treat   the   mixture  by  the   antiformin   method 
(vide  Appendix,  page  502). 

3.  Inoculate    each    of    two    guinea-pigs,    intraperi- 
toneally,  with  half  of  the  emulsion  thus  obtained. 

4.  Kill  one  of  the  guinea-pigs  at  the  end  of  the  first 
week  and  examine  carefully. 

5 .  Kill  the  second  guinea-pig  at  the  end  of  the  second 
week  and  examine  carefully. 

6.  Utilise  the  remainder  of  the  deposit  for  micro- 
scopical examination  and  cultivations  upon  Dorsett's 
egg  medium. 

NOTE. — No  value  whatever  attaches  to  the  result  of  a  micro- 
scopical examination  for  the  presence  of  the  B.  tuberculosis 
unless  confirmed  by  the  result  of  inoculation  experiments. 

6.  Streptococcus  Pyogenes  Longus.— 

(A)  i.  Spread  serial  surf  ace  plates  upon  nutrose  agar. 
Also  plant  serial  cultivations  upon  sloped  nutrient  agar 
(six  tubes  in  series) . 

2.  If  the  resulting  growth  shows  colonies  which  resem- 
ble those  of  the  streptococcus,  make  subcultivations 
upon  agar  and  in  bouillon,  in  the  first  instance,  and 
study  carefully. 

(B)  i.  Plant  a  large  loopful  of  the  deposit  D2  into 
each  of  three  tubes  of  glucose  formate  bouillon,  and 
incubate  anaerobically  (in  Buchner's  tubes)  for  twenty- 
four  hours  at  3  7°  C. 


456  BACTERIOLOGICAL  ANALYSES 

2.  If  the  resulting  growth  resembles  that   of  the 
streptococcus,  make  subcultivations  upon  nutrient  agar. 

3 .  Prepare  subcultivations  of  any  suspicious  colonies 
that  appear,  upon  all  the  ordinary  media,  and  study 
carefully. 

If  the  streptococcus  is  successfully  isolated,  inocu- 
late serum  bouillon  cultivations  into  the  mouse,  guinea- 
pig,  and  rabbit,  to  determine  its  pathogenicity  and 
virulence. 

7.  Staphylococcus  Pyogenes  Aureus. — 

1.  Examine  carefully  the  growth  upon  the  serial 
blood   serum  cultivations  prepared  to  isolate  B.  diph- 
theriae  and  the  serial  agar  cultivations  to  isolate  strep- 
tococci after  forty-eight  hours'  incubation. 

2.  Pick  off  any  suspicious  orange  coloured  colonies, 
plant  on  sloped  agar,  and  incubate  at  20°  C.     Observe 
pigment  formation. 

3.  Prepare    subcultivations    from    any    suspicious 
growths  upon  all  the  ordinary  media,  study  carefully 
and  investigate  their  pathogenicity. 

8.  Micrococcus  Melitensis. — The  milk  from  an  animal 
infected  with  M.  melitensis  usually  contains  the  organ- 
isms in  large  numbers  and  but  few  other  bacteria. 

1.  Spread  several  sets  of  surface  plates  upon  nutrose 
agar,  each  from  one  loopful  of  the  deposit  in  tube  D1 
orD2. 

2 .  Spread  several  sets  of  surface  plates  upon  nutrose 
agar,  each  from  one  drop  of  the  original  milk  sample. 

3.  Incubate  aerobically  at  37°  C.  and  examine  daily 
up  to  the  end  of  ten  days. 

4.  Pick  off  suspicious  colonies,  examine  them  micro- 
scopically and  subcultivate  upon  nutrose  agar  in  tubes ; 
upon  glucose  agar  and  in  litmus  milk. 

5.  Test  the  subsequent  growth  against  the  serum  of 
an  experimental  animal  inoculated  against  M.  melitensis 
to  determine  its  agglutinability. 


ICE  CREAM:  BUTTER  457 

6.  If  apparently  M.  melitensis,  inoculate  growth  from 
a  nutrose  agar  culture  after  three  days  incubation  in- 
tracranially  into  the  guinea-pig. 

ICE  CREAM. 

Collection  of  the  Sample.— 

1.  Remove  the  sample  from  the  drum  in  the  ladle 
or  spoon  with  which  the  vendor  retails  the  ice  cream, 
and  place  it  at  once  in  a  sterile  copper  capsule,  similar 
to  that  employed  for  earth  samples  (vide  page  471). 

2.  Pack  for  transmission  in  the  ice-box. 

3.  On  arrival  at  the  laboratory  place  the  copper 
capsules  containing  the  ice  cream  in  the  incubator  at 
20°  C.  for  fifteen  minutes — that  is,  until  at  least  some 
of  the  ice  cream  has  become  liquid. 

Qualitative  and  Quantitative  Examination. — Treat 
the  fluid  ice  cream  as  milk  and  conduct  the  examina- 
tion in  precisely  the  same  manner  as  described  for 
milk  (vide  page  443). 

EXAMINATION  OF  CREAM  AND  BUTTER. 

Collection  of  the  Sample. — Collect,  store,  and  trans- 
mit samples  to  the  laboratory,  precisely  as  is  done  in 
the  case  of  ice  cream. 

Quantitative. — 

Apparatus  Required: 

Sterile  test-tube. 

Sterilised  spatula. 

Water-bath  regulated  at  42°  C. 

Case  of  sterile  plates. 

Case  of  sterile  graduated  pipettes,  i  c.c.  (in  hundredths). 

Tubes  of  gelatine-agar  (+  10  reaction). 

Plate-levelling  stand,  with  its  water  chamber  filled  with  water 
at  42°  C. 

METHOD.— 

i.  Transfer  a  few  grammes  of  the  sample  to  a  sterile 
test-tube  by  means  of  the  sterilised  spatula. 


458  BACTERIOLOGICAL  ANALYSES 

2.  Place  the  tube  in  the  water-bath  at  42°  C.  until 
the  contents  are  liquid. 

3.  Liquefy  eight  tubes  of  gelatine-agar  and  place 
them  in  the  water-bath  at  42°  C.,  and  cool  down  to 
that  temperature. 

4.  Inoculate  the  gelatine-agar  tubes  with  the  fol- 
lowing quantities  of  the  sample  by  the  help  of  a  sterile 
pipette  graduated  to  hundredths  of  a  cubic  centimetre 
—viz., 

To  tube  No.  i  add  i         c.c.  liquefied  butter. 

2  add  o .  5     c.c.  liquefied  butter. 

3  add  o .  3     c.c.  liquefied  butter.     . 

4  add  o .  2     c.c.  liquefied  butter. 

5  add  o .  i     c.c.  liquefied  butter. 

6  add  0.05  c.c.  liquefied  butter. 

7  add  0.03  c.c.  liquefied  butter. 

8  add  0.02  c.c.  liquefied  butter. 

9  add  o.oi  c.c.  liquefied  butter. 

5.  Pour  a  plate  cultivation  from  each  of  the  gelatine- 
agar  tubes  and  incubate  at  28°  C. 

6.  "Count"  the  plates  after  three  days'  incubation, 
and  from  the  figures  thus  obtained  estimate  the  number 
of  organisms  present  per  cubic  centimetre  of  the  sample. 

Qualitative. — 

Apparatus  Required: 

Sterile  beaker,  its  mouth  plugged  with  sterile  cotton-wool. 

Counterpoise  for  beaker. 

Scales  and  weights. 

Sterilised  spatula. 

Water-bath  regulated  at  42°  C. 

Separatory  funnel,  250  c.c.  capacity,  its  delivery  tube  protected 
against  contamination  by  passing  it  through  a  cotton-wool 
plug  into  the  interior  of  a  small  Erlenmeyer  flask  which  serves 
to  support  the  funnel.  This  piece  of  apparatus  is  sterilised 
en  masse  in  the  hot-air  oven. 

Large  centrifugal  machine. 

Sterile  tubes  (for  the  centrifuge)  closed  with  solid  rubber 
Stoppers. 


BUTTER  459 

Case  of  sterile  pipettes,  10  c.c. 

Case  of  sterile  graduated  pipettes,  i  c.c.  (in  tenths  of  a  cubic 
centimetre) . 

METHOD. — 

1.  Weigh  out  100  grammes  of  the  sample  in  a  sterile 
beaker. 

2.  Plug  the  mouth  of  the  beaker  with  sterile  cotton- 
wool and  immerse  the  beaker  in  a  water-bath  at  42°  C. 
until  the  contents  are  completely  liquefied. 

3 .  Fill  the  liquefied  butter  into  the  sterile  separatory 
funnel. 

4.  Transfer  the  funnel  to  the  incubator  at  37°  C. 
and  allow  it  to  remain  there  for  four  days. 

At  the  end  of  this  time  the  contents  of  the  funnel 
will  have  separated  into  two  distinct  strata. 

(a)  A  superficial  oily  layer,   practically  free  from 
bacteria. 

(b)  A  deep  watery  layer,  turbid  and  cloudy  from 
the  growth  of  bacteria. 

5.  Draw  off  the  subnatant  turbid  layer  into  sterile 
centrifugal  tubes,  previously  warned  to  about  42°  C., 
and  centrifugalise  at  once. 

6.  Pipette  off  the  supernatant  fluid  and  fill  the  tubes 
with   sterile    i    per   cent,    sodium   carbonate   solution 
previously   warmed   slightly;   stopper  the   tubes   and 
shake  vigour ously  for  a  few  minutes. 

7.  Centrifugalise  again. 

8.  Pipette  off  the  supernatant  fluid;  filling  the  tubes 
with  warm  sterile  bouillon,  shake  well,  and  again  centri- 
fugalise, to  wash  the  deposit. 

9.  Pipette  off  the  supernatant  fluid. 

10.  Prepare   cover-slip   preparations,    fix  and   clear 
as  for  milk  preparations,  stain  carbolic  methylene-blue, 
Gram's  method,  Ziehl-Neelsen's  method,  and  examine 
microscopically   with   a  TV    inch    oil-immersion   lens. 

11.  Proceed  with  the  examination  of  the  deposit 
as  in  the  case  of  milk  deposit  (see  pages  450  et  seq.) . 


460  BACTERIOLOGICAL  ANALYSES 

EXAMINATION  OF  UNSOUND  MEATS. 
(INCLUDING  TINNED  OR  POTTED  MEATS,  FISH,  ETC.) 

The  bacterioscopic  examination  of  unsound  food  is 
chiefly  directed  to  the  detection  of  those  members  of 
the  Coli-typhoid  group — B.  enteriditis  of  Gaertnerand 
its  allies — which  are  usually  associated  with  epidemic 
outbreaks  of  food  poisoning,  and  such  anaerobic  bac- 
teria as  initiate  putrefactive  changes  in  the  food  which 
result  in  the  formation  of  poisonous  ptomaines,  conse- 
quently the  quantitative  examination  pure  and  simple 
is  frequently  omitted. 

A.  Cultural  Examination. 
Quantitative. — 

Apparatus  Required: 

Sterilised  tin  opener,  if  (necessary.) 

Erlenmeyer  flask  (500  c.c.  capacity)  containing  200  c.c.  sterile 
bouillon  and  fitted  with  solid  rubber  stopper. 

Counterpoise. 

Scissors  and  forceps. 

Scales  and  weights. 

Water  steriliser. 

Hypodermic  syringe. 

Syringe  with  intragastric  tube. 

Rat  forceps. 

Case  of  sterile  capsules. 

Filtering  apparatus  as  for  water  analysis. 

Case  of  sterile  plates. 

Case  of  sterile  graduated  pipettes,  10  c.c.  (in  tenths  of  a  cubic 
centimetre) . 

Case  of  sterile  graduated  pipettes,  i  c.c.  (in  tenths  of  a  cubic 
centimetre) . 

Plate-levelling  stand. 

Tubes  of  nutrient  gelatine. 

Tubes  of  nutrient  agar. 

Water-bath  regulated  at  42°  C. 

Bulloch's  apparatus. 

METHOD. — 

i.  Place  the  flask  containing  200  c.c.  sterile  broth 
on  one  pan  of  the  scales  and  counterpoise  accurately. 


FOOD  461 

2.  Mince  a  portion  of  the  sample  by  the  aid  of  sterile 
scissors  and  forceps,  and  add  the  minced  sample  to 
the  bouillon  in  the  flask  to  the  extent  of  20  grammes. 

3.  Make  an  extract  by  standing  the  flask  in  the 
incubator  running  at  42°  C.  (or  in  a  water- bath  regu- 
lated to  that  temperature)  for  half  an  hour,  shaking  its 
contents  from  time  to  time.     Better  results  are   ob- 
tained if  an  electrical  shaker  is  fitted  inside  the  incu- 
bator  and   the  flask  kept  in  motion  throughout  the 
entire  thirty  minutes. 

Now  every  centimetre  contains  the  bacteria  washed 
out  from  o.i  gramme  of  the  original  food. 

4.  Inoculate  tubes  of  liquefied  gelatine  as   follows: 

To  tube  No.  i  add  i.o  c.c.  of  the  extract. 

2  add  o.  5  c.c.  of  the  extract. 

3  add  0.3  c.c.  of  the  extract. 

4  add  o.  2  c.c.  of  the  extract. 

5  add  o.i    c.c.  of  the  extract. 

Pour  plates  from  these  tubes  and  incubate  at  20°  C. 

5.  Prepare  a  precisely  similar  set  of  agar  plates  and 
incubate  at  3  7°  C. 

6.  Pipette  5  c.c.  of  the  extract  into  a  sterile  tube, 
heat    in    the   differential  steriliser   at  80°  C.  for  ten 
minutes. 

7.  From  the  heated  extract  prepare  duplicate  sets  of 
agar  and  gelatine  plates  and  incubate  anaerobically 
in  Bulloch'sapparatusat  37°C.  and  20°  C.  respectively. 

8.  After  three  days'  incubation  examine  the  agar 
plates   both   aerobic  and   anaerobic    and    enumerate 
the  colonies  developed  from  spores  (7),  and  from  vege- 
tative forms  and  spores  (5),  and  calculate  and  record 
the  numbers  of  each  group  per  gramme  of  the  original 
food. 

9.  After  seven  days*  incubation  (or  earlier  if  com- 
pelled by  the  growth  of  liquefying  colonies)  enumerate 
the  gelatine  plates  in  the  same  way. 


462  BACTERIOLOGICAL  ANALYSES 

10.  Subcultivate  from  the  colonies  that  make  their 
appearance  and  identify  the  various  organisms. 

1 1 .  Continue  the  investigations  with  reference  to  the 
detection  of  pathogenic  organisms  as  described  under 
water  (page  429  et  seq.). 

Qualitative. — 

I.  Cultural. 

The  micro-organisms  sought  for  during  the  examina- 
tion of  unsound  foods  comprise  the  following : 

Members  of  the  Coli-typhoid  groups  (chiefly  those  of 
the  Gaertner  class). 

B.  anthracis. 

Streptococci 

Anaerobic  Bacteria: 

B.  enteritidis  sporogenes. 
B.  botulinus. 
B.  cadaveris. 

The  methods  by  which  these  organisms  if  present 
may  be  identified  and  isolated  have  already  been 
discribed  under  the  corresponding  section  of  water 
examination  with  the  exception  of  those  applicable  to 
B.  botulinus,  and  B.  cadaveris.  These  can  only  be 
isolated  satisfactorily  from  the  bodies  of  experimen- 
tally inoculated  animals. 

II  Experimental 
Tissue. — 

1.  Feed  rats  and  mice  on  portions  of  the  sample 
and  observe  the  result. 

2.  If  any  of  the  animals  die,  make  complete  post- 
mortem examinations  and  endeavour  to  isolate  the 
pathogenic  organisms. 


OYSTERS  463 


Extract. — 


1.  Introduce  various  quantities  of  the  bouillon  ex- 
tract into  the  stomachs  of  several  rats,  mice  and  guinea- 
pigs  repeatedly  over  a  period  of  two  or  three  days  by 
the  intragastric  method  of  inoculation  (see  page  367) 
and  observe  the  result.     Guinea-pigs  and  mice  are  very 
susceptible  to  infection  by  B.  botulinus  by  this  method; 
rabbits  less  so. 

2.  Inoculate  rats,   mice,    and   guinea-pigs    subcuta- 
neously  into  deep  pockets,  and  intraperitoneally  with 
various  quantities  of  the  bouillon  extract,  and  observe 
the  result. 

3.  Filter  some  of  the  extract  through  a  Chamberland 
candle    and   incubate   the   filtrate   to   determine   the 
presence  of  soluble  toxins. 

4.  If  any  of  the  animals  succumb  to  either  of  these 
methods    of   inoculation,    make    careful    post-mortem 
examinations  and  endeavour  to  isolate  the  pathogenic 
organisms. 

THE   EXAMINATION  OF  OYSTERS  AND   OTHER  SHELLFISH. 

On  opening  the  shell  of  an  oyster  a  certain  amount 
of  fluid  termed  "liquor"  is  found  to  be  present.  This 
varies  in  amount  from  a  drop  to  many  cubic  centi- 
metres (o.i  c.c.  to  10  c.c.) — in  the  latter  case  the  bulk 
of  the  fluid  is  probably  the  last  quantum  of  water  in- 
gested by  the  bivalve  before  closing  its  shell.  In 
order  to  obtain  a  working  average  of  the  bacterio- 
logical flora  of  a  sample,  ten  oysters  should  be  taken 
and  the  body,  gastric  juice  and  liquor  should  be 
thoroughly  mixed  before  examination.  The  examina- 
tion, as  in  dealing  with  other  food  stuffs,  is  directed  to 
the  search  for  members  of  the  Coli-typhoid  group, 
sewage  streptococci  and  perhaps  also  B.  enteritidis 
sporogenes. 


464  BACTERIOLOGICAL  ANALYSES 

Apparatus  Required: 

Two  hard  nail  brushes. 

Liquid  soap. 

Sterile  water  in  aspirator  jar  with  delivery  nozzle  controlled 
by  a  spring  clip. 

Sterile  oyster  knives. 

Sterile  glass  dish,  with  cover,  sufficiently  large  to  accommodate 
ten  oysters. 

Sterile  forceps. 

Sterile  scissors. 

Sterile  towels  or  large  gauze  pads. 

Sterile  graduated  cylinders  1000  c.c.  capacity,  with  either  the 
lid  or  the  bottom  of  a  sterile  Petri  dish  inverted  over  the 
open  mouth  as  a  cover. 

Glass  rods. 

Corrosive  sublimate  solution,  i  per  mille. 

Bile  salt  broth  tubes. 

Litmus  milk  tubes. 

Surface  plates  of  nutrose  agar. 

Case  of  sterile  pipettes,  i  c.c.  (in  tenths  of  a  c.c.) 

Case  of  sterile  pipettes,  10  c.c.  (in  tenths  of  a  c.c.) 

Case  of  sterile  glass  capsules. 

Erlenmeyer  flasks,  250  c.c.  capacity. 

Double  strength  bile  salt  broth. 

METHOD. — 

1.  Thoroughly  clean  the  outside  of  the  oyster  shells 
by  scrubbing  each  in  turn  with  liquid  soap  and  nail 
brush  under  a  tap  of  running  water.     Then,  holding 
an  oyster  shell  in  a  pair  of  sterile  forceps  wash  every 
part  of  the  outside  of  the  shell  with  a  stream  of  sterile 
water  running  from  an  aspirator  jar;  deposit  the  oyster 
inside  the  sterile  glass  dish.     Repeat  the  process  with 
each  of  the  remaining  oysters. 

2.  Before    proceeding    further,    cleanse    the    hands 
thoroughly  with   clean  nail   brush,   soap  and   water, 
then  plunge  them  in  lysol  2  per  cent,  solution,  and, 
finally  in  sterile  water. 

3.  Spread  a  sterile  towel  on  the  bench. 

4.  Remove  one  of  the  oysters  from  the  sterile  glass 
dish  and  place  it,  resting  on  its  convex  shell,  on  the 


OYSTERS  465 

towel.  Turn  a  corner  of  the  sterile  towel  over  the 
upper  flat  shell  to  give  a  firmer  grip  to  the  left  hand, 
which  holds  the  shell  in  position. 

5.  With  the  sterile  oyster  knife  (in  the  right  hand) 
open  the  shell  and  separate  the  body  of  the  oyster 
from  the  inner  surface  of  the  upper  flat  shell.     Bend 
back  and  separate  the  flat  shell,  leaving  the  body  of  the 
oyster  in  and  attached  to  the  concave  shell.     Avoid 
spilling  any  of  the  liquor. 

(Some    dexterity    in    opening    oysters    should    be 
acquired  before  undertaking  these  experiments). 

6.  Cut  up  the  body  of  the  oyster  with  sterile  scissors 
into  small  pieces  and  allow  the  liquor  freed  from  the 
body  during  the  process  to  mix  with  the  liquor  previ- 
ously in  the  shell. 

7.  Transfer  the  comminuted  oyster  and  the  liquor  to 
the  cylinder. 

8.  Treat  each  of  the  remaining  oysters  in  similar 
fashion. 

9.  Mix  the  contents  of  the  cylinder  thoroughly  by 
stirring  with  a  sterile  glass  rod.     The  total  volume  will 
amount  to  about  100  c.c. 

10.  Use  o.i  c.c.  of  the  mixed  liquor  to  inseminate 
each  of  a  series  of  three  nutrose  surface  plates. 

11.  Inoculate  o.i  c.c.  of  the  mixed  liquor  into  each 
of  three  tubes  of  litmus  milk. 

12.  Add  sterile  distilled  water  to  the  contents  of  the 
cylinder  up  to   1000  c.c.  and  stir  thoroughly  with  a 
sterile  glass  rod  and  allow  to  settle.     The  bacterial 
content  of  each  oyster  may  be  regarded,  for  all  practi- 
cal purposes,  as  comprised  in  100  c.c.  of  fluid. 

13.  Arrange  four  glass  capsules  in  a  row  and  number 
I,  II,  III,  IV.     Pipette  9  c.c.  sterile  distilled  water  into 
each. 

14.  To  capsule  No.  I  add  i  c.c.  of  the  diluted  liquor, 
etc.  from  the  cylinder,  and  mix  thoroughly.     To  cap- 
sule II  add  i  c.c.  of  dilution  in  capsule  I  and  mix  thor- 

3° 


466  BACTERIOLOGICAL  ANALYSES 

oughly.  Carry  over  i  c.c.  of  fluid  from  capsule  II  to 
capsule  III,  afterwards  adding  i  c.c.  of  fluid  from  capsule 
III  to  capsule  IV. 

15.  Label  tubes  of  bile  salt  broth  and  inoculate  with 
the  following  amounts  of  diluted  oysters : 

No.  6  with  10  c.c.  cylinder  fluid       =  o.  i  oyster. 

No.  5  with     i  c.c.  cylinder        fluid  =  0.01  oyster. 

No.  4  with     i  c.c.  capsule      I  fluid  =  0.001  oyster. 

No.  3  with     i  c.c.  capsule    II  fluid  =  0.0001  oyster. 

No.  2  with     i  c.c.  capsule  III  fluid  =  0.00001  oyster. 

No.  i  with     i  c.c.  capsule  IV  fluid  =  0.000001  oyster. 

1 6.  Transfer  100  c.c.  cylinder  fluid  (=  i  oyster)  to  an 
Erlenmeyer  flask  and  add  50  c.c.  double  strength  bile 
salt  broth,  and  label  7. 

17.  Duplicate  all  the  above  indicated  cultures. 

1 8.  Put  up  the  tube  cultures  in  Buchner's  tubes  and 
incubate  anaerobically  at  42°  C. 

If  growth  occurs  in  tube  I  the  organism  finally  iso- 
lated, e.g.,  B.  coli,  must  have  been  present  to  the  extent 
of  one  million  per  oyster. 

19.  Complete  the  examination  for  members  of  the 
Coli-typhoid  group  and  sewage  streptococci,  as  directed 
under  Water  Examination,  page  429  (steps  11-21). 

20.  Inoculate  a  series  of  6  tubes  of  litmus  milk  with 
quantities  of  the  material  similar  to  those  indicated  in 
step    15;  heat  to  80°  C.  for  ten  minutes,   and  incu- 
bate under  anaerobic  conditions  at  37°  C.     Examine 
for  the  presence  of  B.  enteritidis  sporogenes  as  directed 
under  Water  Examination,  page  438  (steps  7-10). 


EXAMINATION  OF  SEWAGE  AND  SEWAGE  EFFLUENTS. 

Quantitative.— 

Collection  of  the  Sample. — As  only  small  quantities 
of  material  are  needed,  the  samples  should  be  collected 
in  a  manner  similar  to  that  described  under  water  for 


SEWAGE  467 

quantitative  examination  and  transmitted  in  the  ice 
apparatus  used  in  packing  those  samples. 

Apparatus  Required. — As  for  water  (vide  page  420) . 

METHOD. — 

1.  Arrange  four  sterile  capsules  in  a  row  and  number 
them  I,  II,  III,  IV. 

2.  Pipette  9  c.c.  sterile  bouillon  into  capsule  No.  I. 

3.  Pipette  9.9  c.c.  sterile  bouillon  into  capsules  II, 
III,  and  IV. 

4.  Add  i  c.c.  of  the  sewage  to  capsule  No.  I  by  means 
of  a  sterile  pipette,  and  mix  thoroughly. 

5.  Take   a   fresh   sterile   pipette    and    transfer    o.i 
c.c.    of   the   mixture   from   No.  I  to  No.  II  and  mix 
thoroughly. 

6.  In  like  manner  transfer  o.i  c.c.  from  No.  II  to 
No.  Ill,  and  then  o.i  c.c.  from  No.  Ill  to  No.  IV. 

Now  i  c.c.  of  dilution  No.      I  contains  o .  i  c.c.  of  the  original  sewage, 

i  c.c.  of  dilution  No.     II  contains  o.ooi  c.c.  of  the  original  sewage, 

i  c.c.  of  dilution  No.  Ill  contains  o.ooooi       c.c.  of  the  original  sewage, 
i  c.c.  of  dilution  No.  IV  contains  o.ooooooi  c.c.  of  the  original  sewage. 

7.  Pour  a  set  of  gelatine  plates  from  the  contents 
of  each  capsule,  three  plates  in  a  set,  and  containing 
respectively   0.2,    0.3,    and   0.5    c.c.    of   the   dilution. 
Label    carefully;  incubate  at  20°  C.  for  three,  four,  or 
five  days. 

8.  Enumerate  the  organisms  present  in  those  sets 
of  plates  which  have  not  liquefied,   probably  those 
from  dilution  III  or  IV,  and  calculate  therefrom  the 
number  present  per  cubic  centimetre  of  the  original 
sample  of  sewage. 

Qualitative. — The  qualitative  examination  of  sewage 
is  concerned  with  the  identification  and  enumeration 
of  the  same  bacteria  dealt  with  under  the  corresponding 
section  of  water  examination ;  it  is  consequently  con- 
ducted on  precisely  similar  lines  to  those  already  in- 
dicated (vide  pages  426  to  441). 


468  BACTERIOLOGICAL  ANALYSES 

EXAMINATION  OF  AIR. 

Quantitative.— 

Apparatus  Required: 

Aspirator  bottle,  10  litres  capacity,  fitted  with  a  delivery  tube, 
and  having  its  mouth  closed  by  a  perforated  rubber  stopper, 
through  which  passes  a  short  length  of  glass  tubing. 

Erlenmeyer  flask,  250  c.c.  capacity  (having  a  wide  mouth 
properly  plugged  with  wool),  containing  50  c.c.  sterile  water. 

Rubber  stopper  to  fit  the  mouth  of  the  flask,  perforated  with 
two  holes,  and  fitted  as  follows: 

Take  a  9  cm.  length  of  glass  tubing  and  bend  up  3  cm.  at 
one  end  at  right  angles  to  the  main  length  of  tubing.  Pass 
the  long  arm  of  the  angle  through  one  of  the  perforations  in  the 
stopper;  plug  the  open  end  of  the  short  arm  with  cotton-wool. 

Take  a  glass  funnel  5  or  6  cm.  in  diameter  with  a  stem  12 
cm.  in  length  and  bend  the  stem  close  up  to  the  apex  of  the 
funnel,  in  a  gentle  curve  through  a  quarter  of  a  circle;  pass  the 
long  stem  through  the  other  perforation  in  the  rubber  stopper. 

A  battery  jar  or  a  small  water-bath  to'  hold  the  Erlenmeyer 
flask  when  packed  round  with  ice. 

Supply  of  broken  ice. 

Rubber  tubing. 

Screw  clamps  and  spring  clips,  for  tubing. 

Water  steriliser. 

Retort  stand  and  clamps. 

Apparatus  for  plating  (as  for  enumeration  of  water  organisms, 
vide  page  420). 

METHOD.— 

1.  Fill  10  litres  of  water  into  the  aspirating  bottle 
and  attach  a  piece  of  rubber  tubing  with  a  screw  clamp 
to  the  delivery  tube.     Open  the  taps  fully  and  regulate 
the  screw  clamp,  by  actual  experiment,  so  that  the  tube 
delivers  i  c.c.  of  water  every  second.     The  screw  clamp 
is  not  touched  again  during  the  experiment. 

At  this  rate  the  aspirator  bottle  will  empty  itself 
in  just  under  three  hours.  Shut  off  the  tap  and  make 
up  the  contents  of  the  aspirator  bottle  to  10  litres  again. 

2.  Sterilise  the  fitted  rubber  cork,  with  its  funnel 
and  tubing,  by  boiling  in  the  water  steriliser  for  ten 
minutes. 


AIR 


469 


3.  Remove  the  cotton-wool  plug  from  the  flask,  and 
replace  it  by  the  rubber  stopper  with  its  fittings.     Make 
sure  that  the  end  of  the  stem  of  the  funnel  is  immersed 
in  the  bouillon. 

4.  Place  the  flask  in  a  glass  or  metal  vessel  and  pack 
it  round  with  pounded  ice.     Arrange  the  flask  with  its 
ice  casing  just  above  the  neck  of  the  aspirator  bottle. 


FIG.  216. — Arrangement  of  apparatus  for  air  analysis. 

5.  Connect  up  the  free  end  of  the  glass  tube  from 
the  flask — after  removing  the  cotton-wool  plug — -with 
the   air-entry  tube  in   the  mouth  of  the  aspirating 
bottle  (Fig.  216). 

6.  Open  the  tap  fully,  and  allow  the  water  to  run. 
Replenish  the  ice  from  time  to  time  if  necessary. 

(In  emptying  itself  the  aspirator  bottle  will  aspirate 
10  litres  of  air  slowly  through  the  water  in  the  Erlen- 
meyer  flask.) 

7.  When  the  aspiration  is  completed,  disconnect  the 
flask  and  remove  it  from  its  ice  packing. 


470  BACTERIOLOGICAL   ANALYSES 

8.  Liquefy  three  tubes  of  nutrient  gelatine  and  add 
to  them  0.5  c.c.,  0.3  c.c.,  and  0.2  c.c.,  respectively,  of 
the  water  from  the  flask,  by  means  of  a  sterile  gradu- 
ated pipette,  as  in  the  quantitative  examination  of 
water.     Pour  plates. 

9.  Pour  a  second  similar  set  of  gelatine  plates. 

10.  Incubate  both  sets  of  plates  at  20°  C. 

11.  Enumerate  the  colonies  present  in  the  two  sets 
of  gelatine  plates  after  three,  four,  or  five  days  and 
average  the  results  from  the  numbers  so  obtained; 
estimate  the  number  of  micro-organisms  present  in 
i  c.c.,  and  then  in  the  50  c.c.  of  broth  in  the  flask. 

1 2 .  The  result  of  air  examination  is  usually  expressed 
as  the  number  of  bacteria  present  per  cubic  metre 
(i.  e.,  kilolitre)  of  air;  and  as  the  number  of  organisms 
present  in  the  50  c.c.  water  only  represent  those  con- 
tained in  10  litres  of  air,  the  resulting  figure  must  be 
multiplied  by  100. 

Qualitative.— 

1.  Proceed  exactly  as  in  the  quantitative  examina- 
tion of  air  (vide  supra),  steps  i  to  10. 

2.  Pour  plates  of  wort  agar  with  similar  quantities 
of  the  air-infected  water,  and  incubate  at  37°  C. 

3.  Pour  plates  of  nutrient  agar  with  similar  quan- 
tities of  the  water  and  incubate  at  37°  C. 

4.  Pour  similar  plates  of  wort  gelatine  and  incubate 
at  20°  C. 

5.  Pick  off  the  individual  colonies  that  appear  in 
the  several  plates,  subcultivate  them  on  the  various 
media,  and  identify  them. 

EXAMINATION  OF  SOIL. 

The  bacteriological  examination  of  soil  yields  in- 
formation of  value  to  the  sanitarian  during  the  pro- 
gress of  the  process  of  homogenisation  of  "made  soil" 
(e.  g.,  a  dumping  area  for  the  refuse  of  town)  and 


SOIL  471 

determines  the  period  at  which  such  an  area  may  with 
propriety  and  safety  be  utilised  for  building  purposes; 
or  to  the  agriculturalist  in  informing  him  of  the  suita- 
bility of  any  given  area  for  the  growth  of  crops. 

The  surface  of  the  ground,  exposed  as  it  is  to  the 
bactericidal  influence  of  sunlight  and  to  rapid  alterna- 
tions of  heat  and  cold,  rain  and  wind,  contains  but 
few  micro-organisms.  Again,  owing  to  the  density 
of  the  molecules  of  deep  soil  and  lack  of  aeration  on  the 
one  hand,  and  the  filtering  action  of  the  upper  layers 
of  soil  and  bacterial  antagonism  on  the  other,  bacterial 
life  practically  ceases  at  a  depth  of  about  2  metres. 
The  intermediate  stratum  of  soil,  situated  from  25  to 
50  cm.  below  the  surface,  invariably  yields  the  most 
numerous  and  the  most  varied  bacterial  flora. 

Collection  of  Sample. — A  small  copper  capsule  6  cm. 
high  by  6  cm.  diameter,  with  " pull-off"  cap  secured 
by  a  bayonet  catch,  previously  sterilised  in  the  hot- 
air  oven,  is  the  most  convenient  receptacle  for  samples 
of  soil. 

The  instrument  used  for  the  actual  removal  of  the 
soil  from  its  natural  position  will  vary  according  to 


FIG.  217. — Soil  scoop. 

whether  we  require  surface  samples  or  soil  from  vary- 
ing depths. 

(a)  For  surface  samples,  use  an  iron  scoop,  shaped 
like  a  shoe  horn,  but  provided  with  a  sharp  spine 
(Fig.  217).  This  is  wrapped  in  asbestos  cloth  and 
sterilised  in  the  hot-air  oven.  When  removed  from  the 
oven,  wrap  a  piece  of  oiled  paper,  silk,  or  gutta-percha 
tissue  over  the  asbestos  cloth,  and  secure  it  with 
string,  as  a  further  protection  against  contamination. 


472 


BACTERIOLOGICAL  ANALYSES 


On  reaching  the  spot  whence  the  samples  are  to  be 
taken,  the  coverings  of  the  scoop  are  removed,  and  the 
asbestos  cloth  employed  to  brush  away  loose  stones 
and  debris  from  the  selected  area.  The  surface  soil 
is  then  broken  up  with  the  point  of  the  scoop,  scraped 
up  and  collected  in  the  body  of  the  scoop,  and  trans- 
ferred to  the  sterile  capsule  for  transmission. 


FIG.  218. — Fraenk el's  borer. 

(b)  For  deep  samples  collected  at  various  distances 
from  the  surface,  an  experimental  trench  may  be  cut 
to  the  required  depth  and  samples  collected  at  the 
required  points  on  the  face  of  the  section.  It  is,  how- 
ever, preferable  to  utilise  some  form  of  borer,  such 
as  that  designed  by  Fraenkel  (Fig.  218). 

FraenkeVs  Earth  Borer. — This  instrument  consists 
of  a  stout  hard-steel  rod,  150  cm.  long,  marked  in  centi- 


SOIL  473 

metres  from  the  drill-pointed  extremity.  It  is  pro- 
vided with  a  cross  handle  (adjustable  at  any  point 
along  the  length  of  the  rod  by  means  of  a  screw  nut). 
The  terminal  centimeters  are  thicker  than  the  remainder 
of  the  rod,  and  on  one  side  a  vertical  cavity  about 
0.5  cm.  deep  is  cut.  This  is  covered  by  a  flanged  sleeve 
so  long  as  the  borer  is  driven  into  the  soil  clockwise, 
and  is  opened  for  the  reception  of  the  sample  of  soil, 
when  the  required  depth  is  reached,  by  reversing  the 
screwing  motion,  and  again  closed  before  withdrawal 
of  the  borer  from  the  earth  by  resuming  the  original 
direction  of  twist.  It  can  be  sterilised  in  a  manner 
similar  to  that  adopted  for  the  scoop,  or  by  repeatedly 
rilling  the  cavity  with  ether  and  burning  it  off. 

Quantitative. — Four  distinct  investigations  are  in- 
cluded in  the  complete  quantitative  bacteriological 
examination  of  the  soil : 

1.  The  enumeration  of  the  aerobic  organisms. 

2.  The  enumeration  of  the  spores  of  aerobes. 

3.  The  enumeration  of  the  anaerobic  organisms  (in- 
cluding the  facultative  anaerobes) . 

4.  The  enumeration  of  the  spores  of  anaerobes. 
Further,  by  a  combination  of  the  results  of  the  first 

and  second,  and  of  the  third  and  fourth  of  these,  the 
ratio  of  spores  to  vegetative  forms  is  obtained . 

Apparatus  Required: 

Case  of  sterile  capsules  (25  c.c.  capacity). 

Case  of  sterile  graduated  pipettes,  10  c.c.  (in  tenths  of  a  cubic 
centimetre) . 

Case  of  sterile  graduated  pipettes,  i  c.c.  (in  tenths  of  a  cubic 
centimetre) . 

Flask  containing  250  c.c.  sterile  bouillon. 

Tall  cylinder  containing  2  per  cent,  lysol  solution. 

Plate-levelling  stand. 

12  sterile  plates. 

Tubes  of  nutrient  gelatine. 

Tubes  of  wort  gelatine. 

Tubes  of  nutrient  agar. 

Tubes  of  glucose  formate  gelatine. 


474  BACTERIOLOGICAL  ANALYSES 

Tubes  of  glucose  formate  agar. 
Water-bath  regulated  at  42°  C. 
Bunsen  burner. 
Grease  pencil. 

Sterile  mortar  and  pestle  (agate) . 

Sterile  wide-mouthed  Erlenmeyer  flask  (500  c.c.  capacity). 
Sterile   metal   funnel   with   short   wide   bore   delivery   tube   to 
just  fit  mouth  of  flask. 

Solid  rubber  stopper  to  fit  the  flask  (sterilised  by  boiling) . 

Pair  of  scales. 

Counterpoise  (Fig.  107). 

Sterile  metal  (nickel)  spoon  or  spatula. 

Fractional  steriliser  (Fig.  140). 

METHOD. — 

1.  Arrange  four  sterile  capsules  numbered  I,  II,  III, 
and  IV;  pipette  9  c.c.  sterile  bouillon  into  the  first 
capsule,  and  9.9  c.c.  into  each  of  the  remaining  three. 

2.  Pipette   100  c.c.  sterile  bouillon  into  the  Erlen- 
meyer flask. 

3.  Remove  the  cotton -wool  plug  from  the  flask  and 
replace  it  by  the  sterile  funnel. 

4.  Place  flask  and  funnel  on  one  pan  of  the  scales, 
and  counterpoise  accurately. 

5.  Empty  the  sample  of  soil  into  the  mortar  and 
triturate  thoroughly. 

6.  By  means  of  the  sterile  spatula  add  10  grammes 
of  the  earth  sample  to  the  bouillon  in  the  flask. 

The  final  results  will  be  more  reliable  if  steps  2,  3,  4, 
and  5  are  performed  under  a  hood — to  protect  from 
falling  dust,  etc. 

7.  Remove  the  funnel  from  the  mouth  of  the  flask; 
replace  it  by  the  rubber  stopper  and  shake  vigourously; 
then  allow  the  solid  particles  to  settle  for  about  thirty 
minutes.     One  cubic  centimetre  of  the  turbid*  broth 
contains  the  washings  from  o.i  gramme  of  soil. 

8.  Pipette  off   i   c.c.  of  the  supernatant   bouillon, 
termed  the  "soil  water,"  and  add  it  to  the  contents 
of  capsule  I ;  mix  thoroughly. 

9.  Remove  o.i   c.c.   of  the  infected  bouillon  from 
capsule  I  and  add  it  to  capsule  II,  and  mix. 


SOIL  475 

10.  In  like  manner  add  o.i  c.c.  of  the  contents  of 
capsule  II  to  capsule  III,  and  then  o.i  c.c.  of  the 
contents  of  capsule  III  to  capsule  IV. 

Then  i  c.c.  fluid  from  capsule  I  contains  soil  water  from  .01  gm. 

earth. 
Then  i  c.c.  fluid  from  capsule  II  contains  soil  water  from  .0001 

gm.  earth. 
Then  i    c.c.   fluid  from   capsule   III  contains  soil  water  from 

.000001  gm.  earth. 
Then    i    c.c.   fluid  from   capsule   IV   contains  soil   water  from 

.0000000 1  gm.  earth. 

(A)  Aerobes  (Vegetative  Forms  and  Spores) . — 

IT.  Pour  a  set  of  gelatine  plates  from  the  contents 
of  each  capsule — two  plates  in  a  set,  and  containing 
respectively  o.i  c.c.  and  0.4  c.c.  of  the  diluted  soil 
water.  Label  and  incubate. 

12.  Pour  similar  sets  of  wort  gelatine  plates   from 
the  contents  of  capsules  II  and  III,  label,  and  incubate 
at  20°  C. 

13.  Pour  similar  sets  of  agar  plates  from  the  contents 
of  capsules  II  and  III;  label  and  incubate  at  37°  C. 

14.  Weigh  out  a  second  sample  of  soil — 10  grammes 
—dry  over  a  water-bath  until  of  constant  weight  and 
calculate  the  ratio 

wet  soil  weight 
dry  soil  weight 

15.  "Count"  the  plates  after  incubation  for  three, 
four,  or  five  days,  and  correcting  the  figures  thus  ob- 
tained by  means  of  the  "wet"  to  "dry"  soil  ratio 
estimate — 

(a)  The  number  of  aerobic  micro-organisms  present 
per  gramme  of  the  soil. 

(b)  The  number  of  yeasts  and  moulds  present  per 
gramme  of  the  soil. 

(c)  The  number  of  aerobic  organisms  "growing  at 
37°  C."  present  per  gramme  of  the  soil. 


476  BACTERIOLOGICAL  ANALYSES 

(B)  Anaerobes  (Vegetative  Forms  and  Spores). — 

16.  Pour  similar  sets  of  plates  in  glucose  formate 
gelatine  and  agar  and  incubate  in  Bulloch's  anaerobic 
apparatus. 

(C)  Aerobes  and  Anaerobes  (Spores  Only). — 

17.  Pipette  5  c.c.  soil  water  into  a  sterile  tube. 

1 8.  Place  in  the  differential  steriliser  at  80°  C.  for 
ten  minutes. 

19.  Pour  two  sets  of  four  gelatine  plates  containing 
o.i,  0.2,  0.5,  and  i  c.c.  respectively  of  the  soil  water; 
label  and  incubate  at  20°  C.,  one  set  aerobically,  the 
other  anaerobically  in  Bulloch's  apparatus. 

20.  " Count"  the  plates  (delay  the  enumeration  as 
long  as  possible)  and  estimate  the  number  of  spores 
of    aerobes    and    anaerobes  respectively    present    per 
gramme  of  the  soil. 

21.  Calculate  the  ratio  existing  between  spores  and 
spores  -f  vegetative  forms  under  each  of  the  two  groups, 
aerobic  and  anaerobic  micro-organisms. 

Qualitative  Examination. — The  qualitative  examina- 
tion of  soil  is  usually  directed  to  the  detection  of  one 
or  more  of  the  following : 

Members  of  the  Coli-typhoid  group. 

Streptococci. 

Bacillus  anthracis. 

Bacillus  tetani. 

Bacillus  cedematis  maligni. 

The  nitrous  organisms. 

The  nitric  organisms. 

1.  Transfer   the   remainder   of   the   soil   water    (88 
c.c.)  to  a  sterile  Erlenmeyer  flask  by  means  of  a  sterile 
syphon. 

2.  Fix  up  the  filtering  apparatus  as  for  the  qualita- 
tive examination  of  water,  and  filter  the  soil  water. 

3.  Suspend  the   bacterial  residue  in    5   c.c.   sterile 


SOIL  477 

bouillon  (technique  similar  to  that  described  for  the 
water  sample,  vide  pages  434-436). 

Every  cubic  centimetre  of  suspension  now  contains 
the  soil  water  from  nearly  i  gramme  of  earth. 

The  methods  up  to  this  point  are  identical  no  matter 
which  organism  or  group  of  organisms  it  is  desired 
to  isolate;  but  from  this  stage  onward  the  process  is 
varied  slightly  for  each  particular  bacterium. 

I.  The  Coli=typhoid  Group. — 

II.  Streptococci. — 

III.  Bacillus  Anthracis. — 

IV.  Bacillus  Tetani. — 

The  methods  adopted  for  the  isolation  of  these 
organisms  are  identical  with  those  already  described 
under  water  (page  43  7  et  seq.) . 

V.  Bacillus    (Edematis    Maligni. — Method    precisely 
similar  to  that  employed  for  the  B.  tetani. 

VI.  The  Nitrous  Organisms.— 

1.  Take  ten  tubes  of  Winogradsky's  solution  No  I 
(vide  page  198)  and  number  them  consecutively  from 
i  to  10. 

2.  Inoculate  each  tube  with  varying  quantities  of 
the  material  as  follows : 

To  tube  No.  i  add  i .  o  c.c.  of  the  soil  water. 

To  tube  No.  2  add  o.  i  c.c.  of  the  soil  water. 

To  tube  No.  3  add  i.o  c.c.  from  Capsule  I. 

To  tube  No.  4  add  o .  i  c.c.  from  Capsule  I. 

To  tube  No.  5  add  i.o  c.c.  from  Capsule  II. 

To  tube  No.  6  add  o .  i  c.c.  from  Capsule  II. 

To  tube  No.  7  add  i.o  c.c.  from  Capsule  III. 

To  tube  No.  8  add  o.i  c.c.  from  Capsule  III. 

To  tube  No.  9  add  i.o  c.c.  from  Capsule  IV. 

To  tube  No.  10  add  o.  i  c.c.  from  Capsule  IV. 

Label  and  incubate  at  30°  C. 


478  BACTERIOLOGICAL   ANALYSES 

VII.  The  Nitric  Organisms.— 

3.  Take  ten  tubes  of  Winogradsky's  solution  No  II, 
number  them  consecutively  from  i  to  10  and  inoculate 
with  quantities  of  soil  water  similar  to  those  enum- 
erated in  section  VI  step  2.     Label  and  incubate  at 
30°  C. 

4.  Examine  after  twenty-four  and  forty-eight  hours' 
incubation.     From   those   tubes   that    show   signs    of 
growth   make  subcultivations  in  fresh  tubes  of  the 
same  medium  and  incubate  at  30°  C. 

5.  Make  further  subcultivations  from  such  of  those 
tubes  as  show  growth,  and  again  incubate. 

6.  If    growth    occurs    in    these    subcultures,    make 
surface   smears   on   plates   of   Winogradsky's    silicate 
jelly  (vide  page  198). 

7.  Pick  off  such  colonies  as  make  their  appearance 
and  subcultivate  in  each  of  these  two  media. 

TESTING  FILTERS. 

Porcelain  filter  candles  are  examined  with  reference 
to  their  power  of  holding  back  all  the  micro-organisms 
suspended  in  the  fluids  which  are  filtered  through 
them,  and  permitting  only  the  passage  of  germ-free 
filtrates.  In  order  to  determine  the  freedom  of  the 
filter  from  flaws  and  cracks  which  would  permit  the 
passage  of  bacteria  no  matter  how  perfect  the  general 
structure  of  the  candle  might  be,  the  candle  must  first 
be  attached  by  means  of  a  long  piece  of  pressure  tubing, 
to  a  powerful  pump,  such  as  a  foot  bicycle  pump,  fitted 
with  a  manometer.  The  candle  is  then  immersed  in 
a  jar  of  water  and  held  completely  submerged  whilst 
the  internal  pressure  is  gradually  raised  to  two  atmos- 
pheres by  the  action  of  the  pump.  Any  crack  or  flaw 
will  at  once  become  obvious  by  reason  of  the  stream 
of  air  bubbles  issuing  from  it. 

The  examination  for  permeability  is  conducted  as 
follows : 


FILTERS  479 

Apparatus  Required: 

Filtering  apparatus:  The  actual  filter  candle  that  is  used  must 
be  the  one  it  is  intended  to  test  and  must  be  previously  care- 
fully sterilised;  the  arrangement  of  the  apparatus  will  natur- 
ally vary  with  each  different  form  of  filter,  one  or  other  of  those 
already  described  (vide  pages  42-48). 

Plate-levelling  stand. 

Case  of  sterile  plates. 

Case  of  sterile  pipettes,  10  c.c.  (in  tenths). 

Case  of  sterile  pipettes,  i  c.c.  (in  tenths). 

Tubes  of  nutrient  gelatine. 

Flask  containing  sterile  normal  saline  solution. 

Sterile  measuring  flask,  1000  c.c.  capacity. 

METHOD.— 

1.  Prepare  surface  cultivations,  on  nutrient  agar  in 
a  culture  bottle,  of  the  Bacillus  mycoides,  and  incu- 
bate at  20°  C.,  for  forty-eight  hours. 

2.  Pipette  5  c.c.  sterile  normal  saline  into  the  culture 
bottle  and  emulsify  the  entire  surface  growth  in  it. 

3.  Pipette  the  emulsion  into  the  sterile  measuring 
flask  and  dilute  up  to  1000  c.c.  by  the  addition  of  sterile 
water. 

4.  Pour  the  emulsion  into  the  filter  reservoir  and 
start  the  filtration. 

5.  When  the  nitration  is  completed,  pour  six  agar 
plates  each  containing  i  c.c.  of  the  nitrate. 

6.  Incubate  at  37°  C.  until,  if  necessary,  the  comple- 
tion of  seven  days. 

7.  If  the  filtrate  is  not  sterile,  subcultivate  the  organ- 
ism  passed  and  determine  its  identity  with  the  test 
bacterium  before  rejecting  the  filter — since  the  filtrate 
may  have  been  accidentally  contaminated. 

8.  If  the  filtrate  is  sterile,  resterilise  the  candle  and 
repeat  the  test  now  substituting  a  cultivation  of  B. 
prodigiosus — a  bacillus  of  smaller  size. 

9.  If  the  second  test  is  satisfactory,  test  the  candle 
against   a   cultivation   of   a   very   small  coccus,  e.  g., 
Micrococcus  melitensis,  in  a  similar  manner;   in  this 
instance  continuing  the  incubation  of  cultivations  from 
the  filtrate  for  fourteen  days. 


480  BACTERIOLOGICAL  ANALYSES 

TESTING  OF  DISINFECTANTS. 

Methods  have  already  been  detailed  (page  310)  for 
the  purpose  of  studying  the  vital  resistance  offered  by 
micro-organisms  to  the  lethal  effect  of  germicides.  But 
it  frequently  happens  that  the  bacteriologist  has  to 
determine  the  relative  efficiency  of  " disinfectants" 
from  the  standpoints  of  the  sanitarian  and  commercial 
man  rather  than  from  the  research  worker's  point  of 
view.  In  pursuing  this  line  of  investigation,  it  is  con- 
venient to  compare  the  efficiency,  under  laboratory 
conditions,  of  the  proposed  disinfectant  with  that  of 
some  standard  germicide,  such  as  pure  phenol.  In  so 
doing,  and  in  order  that  the  work  of  different  observers 
may  be  compared,  conditions  as  nearly  uniform  as 
possible  should  be  aimed  at.  The  method  described  is 
one  that  has  been  in  use  by  the  writer  for  many  years 
past,  modified  recently  by  the  adoption  of  some  of 
the  recommendations  of  the  Lancet  Commission  on  the 
Standardisation  of  Disinfectants — particularly  of  the 
calculation  for  determining  the  phenol  coefficient. 

This  method  has  many  points  in  common  with  that 
modification  of  the  "drop"  method  known  as  the 
Rideal- Walker  test. 

General  Considerations. — 

These  may  be  grouped  under  three  headings :  Test 
Germ,  Germicide,  and  Environment. 

i.   Test  Germ. — B.  coli. 

As  disinfectants  are  tested  for  sanitary  purposes,  it  is 
obvious  that  a  member  of  the  coli-typhoid  group  should 
be  selected  as  the  test  germ.  B.  coli  is  selected  on  ac- 
count of  its  relative  nonpathogenicity,  the  ease  with 
which  it  can  be  isolated  and  identified  by  different  ob- 
servers in  various  parts  of  the  world,  the  stability  of  its 
fundamental  characters,  and  evenness  of  its  resistance 
when  utilised  for  these  tests;  finally  since  the  colon 


DISINFECTANTS  481 

bacillus  is  an  organism  which  is  slightly  more  resistant 
to  the  lethal  action  of  germicides  than  the  more  patho- 
genic members  of  this  group,  a  margin  of  safety  is  in- 
troduced into  the  test  which  certainly  enhances  its  value. 
B.  coli  should  be  recently  isolated  from  a  normal 
stool,  and  plated  at  least  twice  to  ensure  the  purity  of 
the  strain;  and  a  stock  agar  culture  prepared  which 
should  be  used  throughout  any  particular  test.  For  any 
particular  experiment  prepare  a  smear  culture  on  agar 
and  incubate  at  3  7°  C.  for  2  4  hours  anaerobically.  Then 
emulsify  the  whole  of  the  surface  growth  in  10  c.c.  of 
sterile  water.  Transfer  the  emulsion  to  a  sterile  test- 
tube  with  some  sterile  glass  beads  and  shake  thoroughly 
to  ensure  homogenous  emulsion.  Transfer  to  a  centri- 
fuge tube  and  centrifugalise  the  emulsion  to  throw 
down  any  masses  of  bacteria  which  may  have  escaped 
the  disintegrating  action  of  the  beads.  Pipette  off  the 
supernatant  emulsion  for  use  in  the  test. 

2.  Germicide. — 

a.  Disinfectant  to  be  tested. — 

The  first  essential  point  is  to  test  the  unknown  disin- 
fectant, which  may  be  referred  to  as  germicide-x,  on 
the  lines  set  out  on  page  3 1 1  to  determine  its  inhibition 
coefficient. 

This  constant  having  been  fixed,  prepare  various  solu- 
tions of  germicide-x  with  sterilised  distilled  water  by  ac- 
curate volumetric  methods,  commencing  with  a  solution 
somewhat  stronger  than  that  representing  the  inhibition 
coefficient.  The  solutions  must  be  prepared  in  fairly 
large  bulk,  not  less  than  5  c.c.  of  the  disinfectant  being 
utilised  for  the  preparation  of  any  given  percentage 
solution. 

b.  Standard  Control— Phenol. 

The  standard  germicide  used  for  comparison  should 
be  one  which  is  not  subject  to  variation  in  its  chemical 
composition,  and  the  one  which  has  obtained  almost 
universal  use  is  Phenol. 


482 


BACTERIOLOGICAL   ANALYSES 


The  following  table  shows  the  effect  of  different  per- 
centages of  carbolic  acid  upon  B.  coli  for  varying  con- 
tact times,  compiled  from  an  experiment  conducted 
under  the  standard  conditions  referred  to  under  Envi- 
ronment. The  results  closely  correspond  to  those 
recorded  by  the  Lancet  Commission  on  Disinfectants, 
1909. 


Percentage  of  phenol 

Contact  time  in  minutes. 

*i 

5 

10 

*5 

20 

25 

3° 

35 

i  20 

+ 
+ 
+ 
+ 
+ 
+ 
+ 

+ 
+ 
+ 
+ 
+ 

+ 

+ 
+ 
+ 

+ 

+ 
+ 

+ 
+ 
+ 

+ 
+ 

+ 

- 

i.io      

i.o    

o.o    .                        

o  8< 

o  80                   

o  <7  r 

w>  /  3  •   • 

0.7  .  .              

06^ 

—  =  No  growth,  i.e.,  bacteria  killed. 
+  =  Growth,  i.e.,  bacteria  still  living. 

From  this  it  will  be  seen  that  the  following  per- 
centage solutions  will  need  to  be  prepared,  namely: 
i.i  per  cent.,  i.o  per  cent.,  0.9  per  cent.,  0.75  per  cent., 
0.7  per  cent.,  as  controls  for  each  experiment. 

Prepare  solutions  of  varying  percentages  by  weighing 
out  the  quantity  of  carbolic  acid  required  for  each  and 
dissolving  in  100  c.c.  of  pure  distilled  water  in  an  ac- 
curately standardised  measuring  flask.  The  solutions 
must  be  prepared  freshly  as  required  each  day. 

Environment. — 

a.  General. — 

Close  the  windows  and  doors  of  the  laboratory  in 
which  the  investigation  is  carried  out,  to  avoid 
draughts.  Flush  over  the  work  bench  and  adjacent 
floor  with  i  :  1000  solution  of  corrosive  sublimate. 


DISINFECTANTS  483 

Caution   the   assistant,  if  one  is  employed,  to  avoid 
unnecessary  movement  or  speech. 

b.  Contact  Temperature,  15-18°  C.— 

This  is  the  temperature  at  which  contact  between  the 
germicide  and  the  test  germ  takes  place,  and  is  of 
importance,  since  some  germicides  (e.  g.,  Phenol)  appear 
to  be  more  powerful  at  high  temperatures.  18°  C. — 
practically  the  ordinary  room  temperature — is  a  tem- 
perature at  which  the  multiplication  of  B.  coli  is  a  com- 
paratively slow  process,  but  variation  of  a  degree  above 
this  temperature  or  of  two  or  three  degrees  below  is  of 
no  moment.  If  the  room  temperature  is  below  15°  C. 
when  the  experiments  are  in  progress,  arrange  a  water- 
bath  regulated  at  18°  C.  for  the  reception  of  the  tubes 
containing  the  mixture  of  germ  and  germicide ;  if  above 
19°  C.  immerse  the  tubes  in  cold  water,  to  which  small 
pieces  of  ice  are  added  from  time  to  time  to  prevent  the 
temperature  rising  above  18°  C. 

c.  Relative    Proportional    Bulk    of    Test    Germ    and 
Germicide,  50  : 1.— 

Five  cubic  centimetres  is  a  convenient  amount  of 
germicidal  solution  to  employ,  and  to  this  o.i  c.c.  of 
the  emulsion  of  test  germ  should  be  added. 

d.  Bulk  of  Sample  Removed  from  Germ -\- Germicide 
Mixture  at  Each  of  the  Time  Periods,  0.1  c.c. — 

This  is  sufficient  to  afford  a  fair  sample  of  the  germ 
content  of  the  mixture,  and  at  the  same  time  is  insuffi- 
cient to  exert  any  inhibitory  action  when  transferred  to 
the  subculture  medium. 

e.  Subculture  Medium.     Bile  Salt  Broth. — 

A  fluid  medium  is  essential  in  order  to  obtain  imme- 
diate dilution  of  the  germicide  carried  over;  at  the  same 
time  it  is  advantageous  to  employ  a  selective  medium 
which  favours  the  growth  of  the  test  germ  to  the 


484  BACTERIOLOGICAL  ANALYSES 

exclusion  of  organisms  likely  to  contaminate  the  prep- 
aration, and  if  possible  one  which  affords  character- 
istic cultural  appearances. 

Bile  Salt  Broth  (page  1 80)  combines  these  desiderata ; 
it  permits  only  the  growth  of  intestinal  bacteria,  whilst 
the  formation  of  an  acid  reaction  and  the  production 
of  gas  in  subcultures  prepared  from  the  germ-germicide 
mixture  is  fairly  complete  evidence  of  the  presence  of 
living  B.  coli. 

The  amount  of  medium  present  in  each  test-tube  is  a 
matter  of  importance,  since  the  medium  not  only  pro- 
vides pabulum  for  the  test  germ,  but  also  acts  as  a 
diluent  to  the  germicide,  to  reduce  its  strength  below 
its  inhibition  coefficient.  For  routine  work  each  sub- 
culture tube  contains  10  c.c.  of  medium,  but  it  is 
obvious  that  if  germicide-x  possesses  an  inhibition 
coefficient  of  o.i  per  cent,  the  addition  of  o.i  c.c.  of 
a  10  per  cent,  solution  to  10  c.c.  of  medium  would  effectu- 
ally prevent  the  subsequent  growth  of  the  test  germ 
after  a  contact  period  insufficient  to  destroy  its  vitality. 
Hence  the  preliminary  tests  may  in  some  instances 
indicate  the  necessity  for  the  presence  of  12  c.c.,  15  c.c. 
or  more  of  the  fluid  medium  in  the  culture  tubes. 

/.  Incubation  Temperature,  37°  C. — 
g.  Observation  Period  of  the  Subcultivations,   Seven 
Days.— 

In  order  to  determine  whether  or  no  the  test  germs 
have  been  destroyed,  observations  must  always  be  con- 
tinued— when  growth  appears  to  be  absent — up  to  the 
end  of  seven  days  before  recording  "no  growth." 

h.  Identification  of  the  Organisms  Developing  in  the 
Subcultivations  after  Contact  in  the  Germ  -f-  Germicide 
Solution. — 

This  is  based  on  the  naked  eye  characters  of  the 
growth  in  the  bile  salt  broth,  supplemented  where 


DISINFECTANTS  485 

necessary  by  plating  methods,  further  subcultivations 
upon  carbohydrate  media  and  agglutination  experi- 
ments. The  sign  (  +  )  is  used  to  indicate  that  growth 
of  the  test  organism  occurred  in  the  subcultivations, 
and  the  sign  (  — )  to  indicate  that  the  test  germs  have 
been  destroyed  and  no  subsequent  growth  has  taken 
place. 

METHOD. — 

Apparatus  Required: 

Sterile  test-tubes  (narrow,  not  exceeding  1.3  cm.  diameter). 

Test-tube  rack  (Fig.  219). 

Sterile  graduated  pipettes  in  case,  i  c.c.  (in  tenths). 

Sterile  graduated  pipettes  in  case,  5  c.  c.(in  c.c.). 

Circular  rubber  washers,  2.5  cm.  diameter  with  central  hole, 
sterilised  by  boiling  immediately  before  use,  then  transferred  to 
sterilised  glass  double  dish. 

Electric  signal  clock  or  stop  watch. 

Sterile  forceps. 

Sterilised  glass  beads. 

Shaking  machine. 

Grease  pencil. 
Material  Required: 

Percentage  solutions  of  germicide-x  (vide  page  481). 

Percentage  solutions  of  pure  phenol  (vide  page  482). 

Aqueous  emulsion  of  B.  coli  (vide  page  481). 

Tubes  of  bile  salt  broth. 

Preliminary  Tests. — 

a.  Inhibition  Coefficient.— 

Determine  the  lowest  percentage  of  germicide-x 
which  inhibits  growth  of  B.  coli  in  the  bile  salt  broth, 
and  the  highest  percentage  which  fails  to  inhibit  (page 
311).  On  the  result  of  this  experiment  determine  the 
bulk  of  medium  required  in  the  subculture  tubes  and 
the  percentage  solutions  to  be  employed  in  the  trial 
trip.  Assuming  the  inhibition  coefficient  to  be  i :  1000, 
it  will  be  quite  safe  to  employ  the  ordinary  culture 
tubes  containing  10  c.c.  medium  in  the  subsequent 
experiments. 


486  BACTERIOLOGICAL  ANALYSES 

b.  Trial  Trip. — 

Determine  the  lethal  effect  of  a  series  of  five  solutions 
of  germicide- x  (say  1:100,  1:250,  1:300,  1:500, 
1:600)  at  contact  times  of  2j,  5,  25  and  30  minutes 
in  the  following  manner: 

1.  Arrange  five  test-tubes  marked  A  to  E  in  the  lower 
tier  of  the  test-tube  rack. 

2.  Into   tube   A  pipette   5   c.c.   germicide-x    1:100 
solution. 

Into  tube  B  pipette  5  c.c.  germicide-x  i  :2oo  solution. 
Into  tube  C  pipette  5  c.c.  germicide-x  i  :3oo  solution. 
Into  tube  D  pipette  5  c.c.  germicide-x  i  :5oo  solution. 
Into  tube  E  pipette  5  c.c.  germicide-x  i  :6oo  solution. 

3 .  Arrange  20  tubes  of  bile  salt  broth  in  the  upper  tier 
of  the  test-tube  rack  in  two  rows,  those  in  the  front 
row  numbered  consecutively  from  left  to  right  i-io, 
those  in  the  back  row  1 1-20. 

4.  Place  a  square  wire  basket  of  about   50  tubes 
capacity  close  to  the  left  of  the  test-tube  rack,  for  the 
reception  of  the  inoculated  tubes. 

5.  Take  a  sterile  i  c.c.  pipette  from  the  case,  pick  up 
a  sterile  rubber  washer  with  forceps  and  push  the  point 
of  the  pipette  into  the  central  hole. 

6.  Put  down  the  forceps  on  the  bench  with  the  sterile 
points  projecting  over  the  edge.     Without  taking  the 
tube  from  the  rack  remove  the  cotton- wool  plug  from 
tube  A,  and  lower  the  pipette,  with  the  rubber  washer 
affixed,  on  to  the  open  mouth  of  the  tube;  with  the 
help  of  the  forceps  to  steady  the  washer,  push  the 
pipette  on  through  the  hole  until  the  point  of  the 
pipette  has  reached  to  within  a  few  millimetres  of  the 
bottom  of  the  tube  (see  fig.  219). 

7.  Adjust  in  the  same  way  a  pipette  and  a  washer  in 
the  mouth  of  each  of  the  other  tubes,  B,  C,  D  and  E. 

8.  Set  the  electric  signal  clock  to  ring  for  the  com- 
mencement of  the  experiment  and  at  subsequent  inter- 
vals of  2j,  5,  25  and  30  minutes. 


DISINFECTANTS  487 

9.  Take  up  0.5  c.c.  of  B.  coli  emulsion  in  sterile 
pipette  graduated  in  tenths  of  a  cubic  centimetre  and 
stand  by. 

10.  As  soon  as  the  bell  rings  lift  the  pipette  from 
tube  A  with  the  left  hand  and  from  the  charged  pipette 
held  in  the  right  hand  deliver  o.i  c.c.  of  B.  coli  emulsion 
into  the  i :  100  solution.     Then  replace  the  pipette  and 
washer. 


FIG.  219. — Test-tube  rack. 

11.  Raise  the  tube  with  the  left  hand  and  shake  it  to 
mix  germ  and  germicide,  whilst  returning  the  delivery 
pipette  in  the  right  hand. 

12.  Repeat  the  process  with  tubes  B,  C,  D  and  E; 
then  drop  the  infected  delivery  pipette  in  the  lysol  jar. 
The  inoculation  of  the  five  tubes  can  be  carried  out 
very  expeditiously,  but  a  period  of  10  seconds  must 
be  allowed  for  each  tube. 

13.  When  the  bell  rings  at  2j  minutes  blow  through 
the  pipette  in  tube  A  (this  agitates  the  germ  +  germicide 
mixture  and  ensures  the  collection  of  a  fair  sample) ; 
allow  the  mixture  to  enter  the  pipette,  and  as  the 
column   of    fluid    extends    well    above   the    terminal 
graduation,    the    right    forefinger    adjusted   over   the 
butt-end  of  the  pipette  before  it  is  lifted  will  retain 


488  BACTERIOLOGICAL  ANALYSES 

more  than  o.  i  c.c.  of  the  mixture  within  the  bore  when 
the  point  of  the  pipette  is  clear  of  the  fluid  in  the  tube. 
Touch  the  point  of  the  pipette  on  the  inner  wall  of  the 
tube,  and  allow  any  excess  of  fluid  to  escape,  only 
retaining  o.i  c.c.  in  the  pipette. 

14.  At  the  same  time,  with  the  left  hand  remove  Bile 
Salt  Tube  No.  i  from  the  upper  tier  of  the  rack,  take 
out  the  cotton-wool  plug  with  the  hand  already  holding 
the  pipette  (the  relative  positions  of  pipette,  plug  and 
culture  tubes  being  practically  the  same  as  those  of 
platinum  loop,  plug  and  culture  tube  shown  in  Fig.  68, 
page  74). 

15.  Insert  the  point  of  the  pipette  into  the  subculture 
tube,  and  blow  out  the  mixture  into  the  medium — re- 
plug the  tube  and  drop  it  into  the  wire  basket.     Re- 
place the  washer-pipette  in  tube  A. 

.  As  soon  as  the  point  of  the  pipette  has  entered  the 
mouth  of  tube  A  it  may  be  released,  since  it  has  already 
been  so  adjusted  that  it  just  clears  the  bottom  of  the 
test-tube,  and  the  elastic  washer  will  prevent  any 
damage  to  the  tube. 

Steps  13,  14  and  15  occupy  on  an  average  10  seconds. 

1 6.  Repeat  steps  13,  14  and  15  with  each  of  the  other 
tubes  B,  C,  D  and  E. 

17.  Repeat  these  various  steps  13-16  when  the  bell 
rings  at  5,  25  and  30  minutes. 

18.  Place  all  the  inoculated  tubes  in  the  incubator 
at  37°  C. 

19.  Examine  the  tubes  at  intervals  of  24  hours,  and 
record  the  results  in  tabular  form  as  shown  in  Table 
page  491  (the  figures  in  the  squares  indicate  the  number 
of  hours  at  which  the  changes  in  the  medium  due  to  the 
growth  of  B.  coli  first  appeared). 

20.  If  a  consideration  of  the  tabulated  results  indi- 
cates strengths  of  Germicide-x  lethal    at  2j  and  30 
minutes  the  final  test  can  be  arranged,   but  if  this 
result  has  not  been  attained,  sufficient  evidence  will 


DISINFECTANTS  489 

probably  be  available  to  enable  a  second  trial  test  to 
be  planned  which  will  give  the  required  information. 

Final  Test. — 

c.     Determination  of  Phenol  Coefficient. — 

X- Disinfectant. — This  comprises  two  distinct  tests, 
one  of  the  Germicide-x,  the  other  of  the  standard 
phenol. 

1 .  Arrange  five  test-tubes  clearly  marked  in  the  lower 
tier  of  the  rack. 

2.  Pipette  into  each  5  c.c.  respectively  of  the  five 
percentage  solutions  of  x-disinfectant  which  the  trial 
run  has  already  shown  will  include  those  affording 
lethal  values  at  2j  and  30  minutes. 

3 .  Arrange  20  tubes  of  bile  salt  broth  in  the  upper  tier 
of  the  test-tube  rack  in  two  rows,  those  in  the  front 
row  numbered  consecutively  from  left  to  right  i-io, 
those  in  the  back  row  11-20. 

4.  Arrange  further  20  tubes  of  bile  salt  broth  num- 
bered 2 1-40  in  two  rows  in  a  second  smaller  rack  which 
can  be  stood  on  the  upper  tier  of  the  rack  as  soon  as 
the  first  20  tubes  have  been  inoculated. 

5.  Place   a   square  wire   basket   of  about    50   tube 
capacity  close  to  the  left  of  the  test-tube  rack,  for  the 
reception  of  the  inoculated  tubes. 

6.  Adjust  a  sterile  i  c.c.  pipette  in  the  mouth  of  each 
of  the  tubes,  A,  B,  C,  D  and  E,  by  means  of  a  washer,  as 
previously  described. 

7.  Set  the  electric  signal  clock  to  ring  for  the  com- 
mencement of  the  experiment  and  subsequently  at 
2^,  5,  10,  15,  20,  25,  30  and  35  minutes. 

8.  Complete  precisely  as  indicated  in   Trial  Runs, 
steps  9-19. 

Control  Phenol. — 

Immediately  the  subculture  tube  from  the  3o-minute 
contact  period  have  been  inoculated,  carry  out  a  pre- 


490  BACTERIOLOGICAL  ANALYSES 

cisely  similar  experiment,  in  which  five  percentage 
strengths  of  Phenol,  (e.  g.,  i.i,  i.o,  0.9,  0.75,  0.7)  are 
arranged  in  the  lower  tier  of  the  test-tube  rack  in 
place  of  the  five  strengths  of  Geimicide-x. 

Calculate  the  phenol   coefficient   by  the   following 
method: 

(a)  Divide  the  figure  representing  the  percentage 
strength  of  the  weakest  lethal  dilution  of  the  carbolic 
acid  control  at  the  2j-minute  contact  period  by  the 
figure   representing    the   percentage    strength    of    the 
weakest  lethal  dilution  of  the  x-disinfectant  at  the 
same  period.     The  quotient  =  phenol  coefficient  at  2j 
minutes. 

(b)  Similarly  obtain   the  phenol   coefficient   at   30 
minutes  contact  period. 

(c)  Record  the  mean  of  the  two  coefficients  obtained 
in  (a)  and  (b)  as  the  mean  phenol  coefficient,  or  simply 
as  the  Phenol  Coefficient. 

The  details  of  the  Final  Test  of  an  actual  determina- 
tion are  set  out  in  the  accompanying  table. 


DISINFECTANTS 


491 


TABLE   27 

Organism  employed,  B.  Coli  Communis. 

Culture  Medium,  Nutrient  Agar  (+  10).    Age,  24  hrs.    Temp,  of  Incubation,  37°  C. 
(  Culture 


Quantities  used     <  ^  >  Emulsion  o.i  c.c.    +5  c.c.  Germicide. 

\  Emulsion  J 

Room  Temperature  during  Experiments,  17°  C. 


Germicide 

Strength 

Time  of  exposure 

Incubation 

a* 

5 

10 

is 

20 

25 

30 

35 

Time 

Temp. 

X 

Germicide-x. 

4% 

— 

— 

— 

— 

— 

— 

— 

— 

7  days. 

37°  C. 

a 
3 
4 

Germicide-x. 

3% 

48 

— 

— 

— 

— 

— 

— 

— 

7  days. 

37°  C. 

Germicide-x. 

2% 

24 

24 

24 

24 

48 

72 

7  days. 

37°  C. 

Germicide-x. 

1% 

24 

24 

24 

24 

72 

24 

72 

7  days. 

37°  C. 

5 

Germicide-x. 

0.5% 

24 

24 

24 

24 

24 

24 

24 

24 

24hours. 

37°  C. 

Phenol  

1.10% 

— 

— 

— 

— 

— 

— 

— 

— 

7  days. 

37°  C. 

2 

3 

Phenol  

1.00% 

24 

7  days. 

37°  C. 

— 

— 

Phenol  

0.75% 

24 

24 

24 

24 

48 

7  days. 

37°  C. 

4 
5 

Phenol  

0.70% 

24 

24 

24 

24 

24 

72 

7  days. 

37°  C. 

Phenol  

0.65% 

24 

24 

24 

24 

24 

48 

24 

2/ 

2  days. 

37°  C. 

Phenol  Coefficient  • 


+ 


0.27+0.35        62 
__^_  =  _  =0.3I 


APPENDIX. 

METRIC  AND  IMPERIAL  SYSTEMS  OF  WEIGHTS  AND 
MEASURES. 

The  initial  unit  of  the  metric  system  is  the  Metre  (m.) 
or  unit  of  length,  representing  one-fourth-millionth 
part  of  the  circumference  of  the  earth  round  the  poles. 

The  unit  of  mass  is  the  Gramme  (g.)>  and  repre- 
sents the  weight  of  one  cubic  centimetre  of  water  at 
its  maximum  density  (viz.  4°  C.  and  760  mm.  mercury 
pressure) . 

The  unit  of  the  measure  of  capacity  is  the  Litre  (/.) , 
and  represents  the  volume  of  a  kilogramme  of  distilled 
water  at  its  maximum  density. 

The  decimal  subdivisions  of  each  of  the  units  are 
designated  by  the  Latin  prefixes '  milli  —  -roVo"  >  centi= 
•J-J--0- ;  deci  =  -^ ;  the  multiples  of  each  unit  .  by  the 
Greek  prefixes  deka=  10;  hecto=  100;  kilo=  1000;  myria 
— 10,000. 

For  a  comparison  of  the  values  of  some  of  the  more 
frequently  employed  expressions  of  the  Metric  System 
and  the  Imperial  System,  the  following  may  be  found 
convenient  for  reference : 

Length : 

i  millimetre  (=  i  mm.)  —-fa  of  an  inch. 

A  centimetre  (=  i  cm.)  =  f  of  an  inch. 

i  inch  (i")  =25  millimetres  or  z\  centimetres. 

Mass: 

i  milligramme  (  =  i  mg.)  =  0.01543  grain  (or  approximately 
&  grain). 

i  gramme  (=  i  g.)  =  15.4323  grains. 

i  "kilo"  or  kilogramme  (=i  kgm.)  =  2  pounds,  3^  ounces 
avoirdupois. 

i  pound  avoirdupois  (  =  i  Ib.)  =453.592  grammes. 

i  ounce  avoirdupois  (  =  i  oz.)=28.35  grammes. 

i  grain  =  0.0648  gramme  or  64:8  milligrammes. 

492 


APPENDIX  493 

Capacity : 

i  cubic  centimetre  (=i  c.c.)  =  i6.9  minims  imperial  measure. 

i  litre  (=i  /.)  =35.196  fluid  ounces  imperial  measure. 

i  fluid  ounec  imperial  measure (=  i  g  )  =  28.42  cubic  centimetres. 

i  pint  imperial  measure(— lO.)  =  568.34  cubic  centimetres. 

i  gallon  imperial  measure  (  =  iC.)=4-546  litres,  or  10  pounds 
avoirdupois,  of  pure  water  at  62°  F.  and  under  an  atmospheric 
pressure  of  30  inches  of  mercury. 

FACTORS    FOR   CONVERTING   FROM   ONE    SYSTEM    TO   THE  OTHER. 

To  convert  grammes  into  grains X  15. 43 2. 

To  convert  grammes  into  ounces  avoirdupois  X    0.03527. 

To  convert  kilogrammes  into  pounds      .    .    .  X    2 . 2046. 
To    convert     cubic    centimetres    into    fluid 

ounces  imperial X    0.0352. 

To  convert  litres  into  fluid  ounces  imperial      .  X  3  5  .  2 . 

To  convert  metres  into  inches X39-37- 

To  convert  grains  into  grammes X    o .  0648. 

To  convert  avoirdupois  ounces  into  grammes .  X  2  8  .  3  5 . 
To  convert  troy  ounces  into  grammes    .    .    .  X3i .  104. 
To    convert   fluid    ounces   into    cubic    centi- 
metres         X28.42. 

To  convert  pints  into  litres       X    0.568. 

To  convert  inches  into  metres X    0.0254. 


494 


APPENDIX 


TABLE  FOR  THE  CONVERSION  OF  DEGREES  CENTI- 
GRADE INTO  DEGREES  FAHRENHEIT. 


Cent. 

Faht. 

Cent. 

'  1 
Faht.     Cent. 

Faht. 

o 

32.0 

34 

93-2 

68 

154.4 

i 

33-8 

35 

95.0 

69 

156.2 

2 

35-6 

36 

96.8 

70 

158.0 

3 

37-4 

37 

98.6 

71 

159.8 

4 

39-2 

38 

100.4 

72 

161.6 

5 

41.0 

39 

102  .  2 

73 

163.4 

6 

42.8 

40 

104.  o 

74 

165  .  2 

7 

44.6 

4i 

105.8 

75 

167.0 

8 

46.4 

42 

107  .  6 

76 

1  68.  8 

9 

48.2 

43 

109.4 

77 

170.  6 

10 

50.  o 

44 

I  I  I  .  2 

78 

172.4 

ii 

51  .8 

45 

II3.O 

79 

174.2 

12 

53-6 

46 

II4-8 

80 

176.0 

13 

55-4 

47 

II6.6 

81 

177.8 

14 

57-2 

48 

II8.4 

82 

179.6 

15 

59-° 

49 

120.  2 

83 

181.4 

16 

60.8 

5° 

122  .0 

84 

183.2 

17 

62.6 

51 

123.8 

85 

185.0 

18 

64.4 

52 

125  .  6 

86 

186.8 

*9 

66.2 

53 

127.4 

87 

188.6 

20 

68.0 

54 

129.2 

88 

190.4 

21 

69.8 

55 

131.0 

89 

192  .  2 

22 

71.6 

56 

132.8 

90 

194.0 

23 

73-4 

57 

134.6 

91 

195.8 

24 

75-2 

58 

136.4 

92 

197.6 

25 

77.0 

59 

138.  2 

93 

199.4 

26 

78.8 

60 

140.  o 

94 

2OI  .  2 

27 

80.6 

61 

141  .8 

95 

203  .  o 

28 

82.4 

62 

143.6 

96 

204  .  8 

29 

84.2 

63 

145-4 

97 

206.  6 

3° 

86.0 

64 

147.2 

98 

208.4 

31 

87.8 

65 

149.0 

99 

210.2 

32 

89.6 

66 

150.8 

IOO 

212  .  O 

33 

91.4 

67 

152.6 

APPENDIX 


495 


TABLE  FOR  THE  CONVERSION  OF  DEGREES  FAHREN- 
HEIT INTO  DEGREES  CENTIGRADE. 


yo 

F  —  ' 

<x  —  $2)  c 

c 

yv 

9 

Lx  » 

. 

. 

. 

1 

fe 

1 

1 

O 

1 

| 

O 

fa 

1 

,c| 
rt 
fe 

i 

32 

0  . 

69 

20  .  6 

105 

40.  6 

141 

60.6 

I77 

80.6 

33 

0.6 

70 

21  .  I 

106 

41.  i 

142 

61.1 

I78 

81.1 

34 

i  .  i 

71 

21.7 

107 

41.7 

143 

61.7 

179 

81.7 

35 

1.7 

72 

22.2 

108 

42.2 

144 

62.2 

180 

82.2 

36 

2  .  2 

73 

22.8 

109 

42.8 

145 

62.8 

181 

82.8 

37 

2.8 

74 

23-3 

I  10 

43-3 

146 

63-3 

182 

83-3 

38 

3-3 

75 

23-9 

iii 

43-9 

147 

63.9 

183 

83-9 

39 

3-9 

76 

24.4 

112 

44.4 

148 

64.4 

184 

84.4 

40 

4.4 

77 

25.0 

113 

45.0 

149 

65  .  o 

185 

85.0 

41 

5-° 

78 

25-6 

114 

45-6 

150 

65.6 

186 

85.6 

42 

5-6 

79 

26.1 

"5 

46  .  i 

151 

66.1 

187 

86.1 

43 

6.1 

80 

26.7 

116 

46.7 

152 

66.7 

188 

86.7 

44 

6.7 

8  1 

27.2 

117 

47.2 

153 

67.2 

189 

87.2 

45 

7.2 

82 

27.8 

118 

47-8 

154 

67.8 

190 

87.8 

46 

7-8 

83 

28.3 

119 

48.3 

155 

68.3 

191 

88.3 

47 

8-3 

84 

28.9 

120 

48.9 

156 

68.9 

192 

88.9 

48 

8.9 

85 

29.4 

121 

49.4 

157 

69.4 

193 

89.4 

49 

9-4 

86 

30.0 

122 

50  .  o 

158 

70.0 

194 

90.  o 

5° 

10.  0 

87 

30.  6 

I23 

5°  •  6 

J59 

70.  6 

*95 

90.  6 

51 

10.  6 

88 

31  .  i 

124 

51-1 

160 

71.1 

196 

91.1 

52 

ii  .  i 

89 

3!-7 

125 

51.7 

161 

71.7 

197 

91.7 

53 

11.7 

90 

32.2 

126 

52.2 

162 

72.2 

198 

92  .  2 

54 

12  .  2 

91 

32.8 

127 

52.8 

163 

72.8 

199 

92.8 

55 

12.8 

92 

33-3 

128 

53-3 

164 

73-3 

200 

93-3 

56 

13-3 

93 

33-9 

I29 

53-9 

165 

73-9 

2OI 

93-9 

57 

13  .9 

94 

34-4 

I30 

54-4 

166 

74-4 

2O2 

94.4 

58 

14.4 

95 

35-° 

131 

55-Q 

167 

75.0 

203 

95-° 

59 

15-° 

96 

35-6 

132 

55-6 

168 

75-6 

2O4 

95-6 

60 

15-6 

97 

36.1 

133 

56.1 

169 

76.1 

205 

96.1 

61 

16.1 

98 

36.7 

134 

56-7 

170 

76.7 

2O6 

96.7 

62 

16.7 

99 

37-2 

135 

57-2 

171 

77-2 

207 

97.2 

63 

17.2 

IOO 

37-8 

I36 

57-8 

172 

77-8 

208 

97.8 

64 

17.8 

101 

38-3 

137 

58.3 

173 

78.3 

209 

98-3 

65 

18.3 

102 

38.9 

138 

58-9 

174 

78.9 

210 

98.9 

66 

18.9 

I03 

39-4 

139 

59-4 

T75 

79-4 

211 

99-4 

67 

19.4 

104 

40.  o 

I40 

60.  o 

176 

80.0 

212 

100.0 

68 

20.  o 

4Q6 


APPENDIX 


Percentage  Formula  for  addition  of  salts,  etc.,  to  completed 
media. 

Formula  for  preparing  any  desired  percentage  of  a  given  salt, 
etc.,  in  tubed  media;  e.  g.,  to  make  4  per  cent,  solution  of  KNO3 
in  a  series  of  tubes  of  broth  each  containing  10  c.c.  of  medium, 
when  there  is  already  available  a  25  per  cent,  stock  aqueous 
solution  of  potassium  nitrate. 

A  (X) 


N  =  number  of  cubic  centimetres  contained  in  each  tube. 
X  =  amount  of  stock  solution  to  be  added  to  each  tube. 
Y  —  percentage  required  in  the  medium. 
A  =  percentage  of  stock  solution. 
Then 

(ip  +  X)  4==g-5X 

IOO  IOO 

Therefore,  40  +  4X  =  2  5X. 

Therefore,         2iX  =  40. 

X=  i .  9  c.c. 
This  allows  for  solution  added  to  the  original  bulk  of  medium. 

Therefore,  10  c.c.  broth +1.9  c.c.  of  a  25  per  cent,  aqueous 
solution  KNO3  makes  11.9  c.c.  medium  containing  4  per  cent. 
KN03. 

TABLES  FOR  PREPARING  DILUTIONS 

(of  Serum,  Disinfectants  or  other  substances.) 
In  estimating  the  agglutinin  content  or  litre  of  a  serum, 
testing  disinfectants  and  for  many  other  purposes,  it  becomes 
necessary  to  prepare  a  series  of  dilutions  of  the  material  under 
examination,  and  in  order  to  avoid  unnecessary  expenditure  of 
labour  it  is  convenient  to  adhere  to  some  definite  scale  of  incre- 
ment, such  for  example  as  the  following : 


From  dilutions  of  i : 

of  5- 

From  dilutions  of  i  : 
of  10. 

From  dilutions  of  i . 
of  25. 

From  dilutions  of  i : 
of  50. 

From  dilutions  of  i : 
of  TOO. 

From  dilutions  of  i  : 
of  250. 

From  dilutions  of  i : 
of  1000. 


10  to  i:  80  rise  by  increments 

80  to  i :  200  rise  by  increments 

200  to  i :  400  rise  by  increments 

400  to  i :  500  rise  by  increments 

500  to  i:  i ooo  rise  by  increments 

1000  to  i :  500°  rise  by  increments 

5000  to  i :  10,000  rise  by  increments 


APPENDIX  497 

From  dilutions  of  i :  10,000  to  i :  100,000  rise  by  increments 
of  5000. 

From  dilutions  of  i :  100,000  to  i :  1,000,000  rise  by  increments 
of  100,000. 

When  dealing  with  a  substance  of  unknown  powers — and  this 
is  especially  true  with  regard  to  agglutinating  sera — it  is  customary 
to  run  a  preliminary  test,  using  a  few  widely  separated  dilutions 
such  as  may  be  obtained  in  the  following  manner: 

FIRST  DILUTION — I. 

i  c.c.  serum  +  9  c.c.  normal  saline  solution  =  10  per  cent, 
solution  or  i:  10  dilution  (of  which  i  c.c.  contains  o.i  c.c.  of  the 
original  serum). 

When  dealing  with  fluids  other  than  serum  the  diluent  is 
usually  distilled  water;  whilst  if  the  original  substance  is  a  solid 
the  instructions  would  read: 

i  gram  o.s.  +  10  c.c.  distilled  water  =  10  per  cent,  solution,  etc. 

SECOND  DILUTION — II. 

i  c.c.  first  dilution  +  9  c.c.  normal  saline  solution  =  i  per 
cent,  solution  or  i :  100  dilution. 

THIRD  DILUTION — III. 

i  c.c.  second  dilution  +  9  c.c.  normal  saline  solution  =  i  per 
mille  solution  or  i:  1000  dilution. 

FOURTH  DILUTION — IV. 

i  c.c.  second  dilution  +  9  c.c.  normal  saline  solution  =  o.  i  per 
mille  solution  or  i  :  10,000  dilution. 

The  following  tables  showing  the  secondary  dilutions  that  can 
readily  be  prepared  from  each  of  these  four  primary  dilutions  for 
use  in  the  subsequent  determination  of  the  exact  titre  will  probably 
be  found  of  service  by  those  who  are  not  ready  mathematicians. 


498 


APPENDIX 


TABLES  FOR  PRE- 


TABLE   I 

Using  10%  stock  solution 
First 
dilution 


Diluent 


TABLE  II 

Using  i  %  stock  solution 
Second 
dilution 


Diluent 


i  :  10  =  i  c.c. 

+   0  C.C. 

J 

IOO  =  I  C.C. 

+   0   C.C. 

i  :  15  =  i  c.c. 

+  0.5  c.c. 

I 

no  =  i  c.c. 

+   O.I  C.C. 

i  :  20  =  i  c.c. 

+  i.o  c.c. 

I 

120  =  I  C.C. 

+   0.2  C.C. 

i  :  25  =  i  c.c. 

+  1.5  c.c. 

[I 

125  =  i  c.c. 

+  0.25  c.c.] 

i  :  30  =  i  c.c. 

+   2.0  C.C. 

I 

130  =  i  c.c. 

+  0.3  c.c. 

i  :  35  =  i  c.c. 

+  2.5  c.c. 

I 

140  =  i  c.c. 

+  0.4  c.c. 

i  :  40  =  i  c.c. 

+  3-0  c.c. 

I 

150  =  i  c.c. 

+  0.5  c.c. 

i  :  45  =  i  c.c. 

+  3-5  c.c. 

I 

160  =  i  c.c. 

+  0.6  c.c. 

i  :  50  =  i  c.c. 

+  4.0  c.c. 

I 

170  =  i  c.c. 

+  0.7  c.c. 

i  :  55  =  i  c.c. 

+   4-5  C.C. 

[I 

175  =  i  c.c. 

+  0.75  c.c.] 

i  :  60  =  i  c.c. 

+  5.0  c.c. 

I 

180  =  i  c.c. 

+  0.8  c.c. 

i  :  65  =  i  c.c. 

+  5-5  c.c. 

I 

190  =  i  c.c. 

+  0.9  c.c. 

i  :  70  =  i  c.c. 

+  6.0  c.c. 

+     f\  r*  r*  r> 

I 

200  =  I  C.C. 

+   I.O  C.C. 

i  :  75  ==  i  c.c. 
i  :  80  =  i  c.c. 

0.5  c.c. 

+  7-o  c.c. 

I 

200  =  I  C.C. 

+   I.O  C.C. 

i  :  80  =  i  c.c. 

+  7.0  c.c. 

I 

I 

225  ==  i  c.c. 
250  =  i  c.c. 

~f~  1.25  c.c. 
+  1.5  c.c. 

i  :  90  =  i  c.c. 

+  8.0  c.c. 

I 

275  =  I  C.C. 

+   1.75  c.c. 

i  :  100  =  i  c.c. 

+  9.00  c.c. 

I 

300  =  i  c.c. 

+   2.0  C.C. 

i  :  1  10  =  i  c.c. 

+  IO.O  C.7. 

I 

325  =  i  c.c. 

+  2.25  c.c. 

i  :  120  =  i  c.c. 

+  I  I.O  C.C. 

I 

350  =  i  c.c. 

+  2.5  c.c. 

[i  :  125  =  i  c.c. 

+  ii.  5  c.c.] 

I 

375  =  i  c.c. 

+   2.75  c.c. 

i  :  130  =  i  c.c. 

+  12.0  C.C. 

1 

400  =  i  c.c. 

+  3.0  c.c. 

i  i  140  ^  i  c.c. 
i  :  150  ==  i  c.c. 

~T~  13  •  o  c.c. 
+  14.0  c.c. 

J. 

400  =  i  c.c. 

+   3-0  c.c. 

i  :  160  =  i  c.c. 

+  15.0  c.c. 

I 

450  =  i  c.c. 

+   3-5  c.c. 

i  :  170  =  i  c.c. 

+  16.0  c.c. 

I 

500  =  i  c.c. 

+  4.0  c.c. 

[i  :  175  =  i  c.c. 

+  16.5  c.c.] 

i  :  180  =  i  c.c. 

+  17.0  c.c. 

I 

500  =  i  c.c. 

+  4.0  c.c. 

i  :  190  =  i  c.c. 

+  18.0  c.c. 

I 

600  =  i  c.c. 

+  5.0  c.c. 

i  :  200  =  i  c.c. 

+  19.0  c.c. 

I 
[T 

700  =  i  c.c. 

+  6.0  c.c. 

1    f.  -  «  ^  1 

i  :  200  =  i  c.o. 

+  19.0  c.c. 

LI 

I 

750  =  i  c.c. 
800  =  i  c.c. 

°-5  c.t,.j 
+  7.0  c.c. 

i  :  225  =  i  c.c. 

+  21.5  c.c. 

I 

900  =  i  c.c. 

+  8.0  c.c. 

i  :  250  =  i  c.c. 

+  24.0  c.c. 

1 

1000  =  I  C.C. 

+  9.0  c.c. 

i  •  275  ==  i  c.c. 
i  :  300  =  i  c.c. 

~T~  2O.5  C.C. 

+  29.0  c.c. 

I 

1000  =  I  C.C. 

+  9.0  c.c. 

i  :  325  =  i  c.c. 

+  31-5  C.C. 

I 

200O  =  I  C.O. 

+  19.0  c.c. 

i  :  350  =  i  c.c. 

+  34..0  C.C. 

i  :  3000  =  i  c.c. 

+  29.0  c.c. 

i  :  375  =  i  c.c. 

+  36.5  c.c. 

i  :  4000  =  i  c.c. 

+  39.0  c.c. 

i  :  400  =  i  c.c. 

+  39.0  c.c. 

i  :  5000  =  i  c.c. 

+  49.0  c.c. 

i  :  400  =  i  c.c. 

+  39.0  C.C. 

i  :  450  =  i  c.c. 

+  44.5  c;c. 

i  :  500  =  i  c.c. 

+  49.0  c.c. 

APPENDIX 


499 


PARING   DILUTIONS. 


TABLE  III 

TABLE    IV 

Using  o.  i  °/ 

D  stock  solution 

Using  0.01% 

stock  solution 

Third        "1 
dilution    J 

Diluent 

Fourth     1 
dilution    J 

+    Diluent 

i 

IOOO 

= 

I   C.C. 

+       0  C.C. 

i 

IO.OOO 

_ 

i 

c.c.  + 

0  C.C. 

i 

1250 

= 

I    C.C. 

+     0.25  c.c. 

i 

I5,OOO 

— 

i 

c.c.  -f 

0.5  c.c. 

i 

1500 

= 

I   C.C. 

+     0.5  c.c. 

i 

20,OOO 

= 

i 

c.c.  + 

1.0  C.C. 

i 

1750 

— 

I  C.C. 

+     0.75  c.c. 

i 

25,000 

- 

i 

c.c.  + 

1.5  c.c. 

i 

2000 

= 

I   C.C. 

+        1.0  C.C. 

i 

30,000 

= 

i 

c.c.  + 

2.0  C.C. 

i 

2250 

= 

I  C.C. 

+     1.25  c.c. 

i 

35,000 

= 

i 

c.c.  + 

2.5  c.c. 

i 

2500 

= 

I   C.C. 

+      1.5  c.c. 

i 

40,000 

= 

i 

c.c.  + 

3.0  c.c. 

i 

2750 

= 

I   C.C. 

+     1.75  c.c 

i 

45,000 

= 

i 

c.c.  + 

3-5  c.c. 

i 

3000 

= 

I   C.C. 

+        2.0  C.C. 

i 

50,000 

= 

i 

c.c.  + 

4.0  c.c. 

i 

3250 

= 

I   C.C. 

+     2.25  c.c. 

i 

55,ooo 

= 

i 

c.c.  + 

4-5  c.c. 

i 

3500 

= 

I   C.C. 

+     2.5  c.c. 

i 

60,000 

= 

i 

c.c.  + 

5.0  c.c. 

i 

3750 

= 

I  C.C. 

+      2.75  c.c. 

i 

65,000 

— 

i 

c.c.  + 

5-5  c.c. 

i 

4000 

= 

I  C.C. 

+     3.0  c.c. 

i 

70,000 

= 

i 

c.c.  + 

6.0  c.c. 

i 

4250 

= 

I  C.C. 

+     3-25  c.c. 

i 

75,000 

= 

i 

c.c.  + 

6.5  c.c 

i 

4500 

= 

I   C.C. 

+     3-5  c.c. 

i 

80,000 

= 

i 

c.c.  + 

7.0  c.c. 

i 

4750 

= 

I   C.C. 

+     3-45  c.c. 

i 

85.000 

= 

i 

c.c.  + 

7-5  c.c. 

i 

5000 

= 

I   C.C. 

+     4.0  c.c. 

i 

90,000 

= 

i 

c.c.  + 

r»  r»      -4- 

8.0  c.c. 

i 

5000 

£.  _  _ 

= 

I   C.C. 

+     4.0  c.c. 

i 

95,ooo 

100,000 

- 

i 

C.C.    T^ 

c.c.  + 

8.5  c.c. 
9.0  c.c. 

i 

i 

oooo 
7000 

= 

I  C.C. 
I   C.C. 

~l~     5  •  o  c.c. 
+     6.0  c.c. 

i 

100,000 

= 

0. 

I   C.C. 

-f-  0.9  c.c. 

[i 

7500 

= 

I   C.C. 

+     6-Sc.c.] 

i 

200,000 

= 

0 

I   C.C. 

+   1.9  c.c. 

i 

8000 

= 

I    C.C. 

+     7.0  c.c. 

[i 

250,000 

= 

0. 

I   C.C. 

+  2.4  c.c. 

i  :     9000 

= 

I   C.C. 

+     8.0  c.c. 

i 

300,000 

= 

o  . 

I   C.C. 

+  2.9  c.c. 

i  :  10,000 

= 

I   C.C. 

+     9.0  c.c. 

i 

400,000 

= 

o  . 

I   C.C. 

+  3-9  c.c. 

i  :  10,000 

= 

I   C.C. 

+     9.0  c.c. 

5      > 

H~  4*9  c.c. 

i  :  15,000 

— 

I  C.C. 

+   14.0  c.c. 

i 

500,000 

= 

0. 

I    C.C. 

+  4.9  c.c. 

i  :  20,000 

= 

I   C.C. 

+   19.0  c.c. 

i 

600,000 

= 

0 

I   C.C. 

+  5.9  c.c. 

i  :  25,000 

= 

I  C.C. 

+  24.0  c.c. 

i 

700,000 

= 

o 

I   C.C. 

+  6.9  c.c. 

i  :  30,000 

= 

I   C.C. 

+  29.0  c.c. 

[i 

750,000 

= 

0 

I    C.C. 

+  7.4  c.c. 

Q 



1 

i 

900,000 

= 

0. 

I   C.C. 

•     7  •  9  c.c. 
+  8.9  c.c. 

i  :  1,000,000 

= 

0 

I   C.C. 

4-9.9  c.c. 

5oo 


APPENDIX 


TEMPERATURE  PRESSURE  TABLE. 


Temperature 
Centigrade 

Mm.  of  Hg. 

Pounds  per  sq.  in. 
absolute  pressure 

Atmospheres 

98° 

707.  i 

13-7 

0-93 

99° 

733  -1 

14.2 

0.96 

100° 

760  .0 

14.7 

I  .  00 

101° 

787-8 

15-2 

1.03 

102° 

816.0 

15-8 

1.07 

103° 

845.2 

16.3 

I  .  II 

104° 

875.4 

16.9 

1  .  15 

105° 

906.4 

i7-5 

1.19 

106° 

938.3 

18.1 

1.23 

107° 

971.1 

18.8 

1.27 

108° 

1004  .  9 

19.4 

1.32 

109° 

1039.6 

20  .  I 

1.36 

110° 

I075-3 

20.8 

1.41 

in0 

II  12  .  0 

21.5 

1  .46 

112° 

i  149  .  8 

22  .  2 

1.51 

"3° 

1188.6 

22.9 

1.56 

114° 

1228  .4 

23-7 

1.61 

"5° 

1269  .  4 

24.5 

1.67 

116° 

1311.4 

25-3 

1.72 

117° 

1354.6 

26.2 

.1.78 

•118° 

1399.0 

27.0 

i  .84 

119° 

1444.5 

27.9 

i  .  90 

120° 

I491-2 

28.8 

i  .  96 

121° 

1539-2 

29-7 

2  .  O2 

122° 

1588.4 

3°-7 

2  .09 

123° 

1638.9 

31  .  7 

2.15 

124° 

1690  .  7 

32.7 

2  .  22 

125° 

1743-8 

33-7 

2  .29 

APPENDIX 


501 


TABLE  FOR  DESICCATION   AT  LOW  TEMPERA- 
TURES  IN  VACUO. 


Temperature 
Centigrade 

Mm.  of  Hg. 

21° 

18.4 

22° 

19  .  6 

23° 

20.8 

24° 

22  .  I 

25° 

23-5 

26° 

24.9 

27° 

26.4 

28° 

28.0 

29° 

29.7 

30° 

31.5 

3i° 

33-3 

32° 

35-3 

33° 

37-3 

34° 

39-5 

35° 

41.7 

36° 

44-1  ' 

37° 

46.6 

38° 

49-2 

39° 

51-9 

40° 

54-8 

4i° 

57.8 

42° 

61.0 

43° 

64.3 

44° 

67-7 

45° 

7i.3 

46° 

75-1 

47° 

79.0 

48° 

83-1 

49° 

87-4 

50° 

91.9 

502  APPENDIX 

ANTIFORMIN  METHOD 

For  the  detection  of  B.  Tuberculosis. 

Antiformin  was  introduced  into  bacteriological 
technique  by  Uhlenhuth  in  1908  for  the  purpose  of 
demonstrating  tubercle  bacilli  when  present  in  small 
numbers,  in  sputum  or  other  material.  It  is  a 
powerful  oxidising  agent  and  rapidly  destroys  most 
bacteria,  but  tubercle  and  other  acid-fast  organisms 
resist  its  lethal  action  for  considerable  periods,  and 
upon  this  fact  the  method  is  based. 

To  prepare  Antiformin  measure  out  and  mix  :— 

Eau  de  Javelle  (Liquor  sodse  chlorinatae  —  B.P.)  50  c.c. 
Sodic  hydrate  15  per  cent,  aqueous  solution  ....  50  c.c. 

METHOD. 

1.  Introduce  the  sputum   or   other   material    (e.g. 
milk  deposit  and  cream ;  pus ;  minced  gland  or  other 
organ;  caseous  material;  broken  down  foci,  etc.)  into 
a  sterile  tube  and  then  add  an  equal  volume  of  anti- 
formin. 

2.  Close  the  tube  with  a  rubber  cork  and  shake 
vigorously    (a   sample  of   antiformin   that   does   not 
"foam"  at  this  stage  is  of  little  use).     Disintegration 
of  the  material  at  once  starts,  associated  bacteria  are 
destroyed  and  the  mixture  rapidly  becomes  a  homo- 
genous but  turbid  fluid — a  process   which   may   be 
hastened  by: — 

3.  Placing  the  tube  in  the  incubator  at  37°  C.  for  30 
minutes — shaking  from  time  to  time. 

4.  Centrifugalise    the    fluid    thoroughly,    at    high 
speed. 

5.  Pipette  off  the  supernatant  fluid,    fill  up    with 
sterile  distilled  water,  cork  the  tube  and  shake  to  dis- 
tribute  the   deposit   throughout    the    water.     Again 
Centrifugalise. 

6.  Repeat  steps  4  and  5  twice  more. 


APPENDIX 


503 


7.  Employ  one  portion  of  the  final  deposit  to  inoc- 
ulate guinea  pigs. 

8.  Plant   the   remainder  of   the  deposit  freely   on 
Dorset's  Egg  medium;  cap  and  incubate  at  37°  C. 

NOTE. — If  only  microscopical  films  are  needed,  fill  up  the  centri- 
fuge tube  with  Ligroin  (a  petroleum  ether)  in  place  of  sterile  dis- 
tilled water  in  step  5  and  prepare  the  films  from  the  surface  of  the 
fluid  to  stain  by  the  Ziehl-Neelsen  process. 


INDEX 


ABBA'S  condenser,  7 
Abbott's  stain  for  spores,  107 
Aberration,  chromatic,  56 

spherical,  55 
Absolute  alcohol  as  a  fixative,  82 

as  an  antiseptic,  27 
Absorbent  paper  for  drying  cover- 
slips,  69 

A.  C.  E.  mixture,  345 
Acetic  acid  for  clearing  films,  82 
Achromatic  condenser,  54 
Acid  haematin,  96 

production,  analysis  table,  283 
by  bacteria,  145 
investigation  of,  280 
qualitative  examination,  283, 

284 

quantitative  examination,  2 80 
Acid-fast    bacilli    in    tissues,    to 

stain,  124 

Action  of  various  gases  on  bac- 
teria, 295 

Active     immunisation,     illustra- 
tive example,  322 
Adjustable  water  bath,  299 
Aerobic  cultures,  221 
Aerogenic  bacteria,  131 
^Esculin  agar,  204 
Agar  gelatine  (guarniari),  194 
methods  of  preparation,  167 
surface  plates,  232 
Agar-agar,  preparation  of,  167 
Agglutination    reaction,    macro- 

scopical,  386 
microscopical,  385 
Agglutinin,  381 
Air,  analysis  of,  468 
filter,  40 

pump,  Geryk,  43 
Albumin  solution,  Mayer's,  120 
Alcohol  production,  test  for,  285 
Alkaline  pyro,  239 
Alum  carmine,  96 
Ammonia  production  test  for,  285 
Amphitrichous  bacteria,  136 
Anaerobic  cultures,  236 
Botkin's  method,  243 
Buchner's  method,  238 
Bulloch's  method,  245 
Hesse's  method,  237 
McLeod's  method,  240 
media,  180 
Novy's  method,  244 


Anaerobic  cultures,  Roux's  bio- 
logical method,  237 
physical  method,  237 
vacuum  method,  238 
Wright's  method,  239 
Anaesthetics,  345 
Analysis  of  air,  apparatus  for,  469 
method  of,  468 
qualitative      bacteriological, 

47Q. 

quantitative        bacteriologi- 
cal, 468 

of  butter,  qualitative  bacterio- 
logical, 458 
quantitative  bacteriological, 

457 

of  cream,  qualitative  bacterio- 
logical, 458 
quantitative  bacteriological, 

457 

of  fish,  460 

of  ice  cream,  qualitative  bac- 
teriological, 457 
of  meat,  apparatus  for,  460 
method  of,  460 
qualitative  bacteriological, 

462 

of  milk,  apparatus  for,  444 
collection  of  samples,  441 
method  of,  441 
qualitative  bacteriological, 

446 
quantitative  bacteriological, 

444 

of  oysters,  463 

of  sewage,  qualitative   bacter- 
iological, 467 
quantitative  bacteriological, 

466 

of  shellfish,  463 
of  soil,  apparatus  for,  473 
collection  of  samples,  471 
method  of,  470 
qualitative      bacteriological, 

476 
quantitative  bacteriological, 

473 
of  water,   apparatus  for,   420, 

427 

collection  of  samples,  416 
method  of,  416 
qualitative  bacteriological, 

426 


505 


5o6 


INDEX 


Analysis   of   water,  quantitative 

bacteriological,  420 
Aniline  dyes,  83 

Gentian  violet,  95 
water,  to  prepare,  108 
Animal  tissue   media    (Frugoni), 

210 

Animals,    natural    infections    of, 

337 

Antiformin  method  for  B.  tuber- 
culosis, 502 

Antigen,  definition  of,  324 

Antiseptics,  27 
action  of,  310 

Apparent  filth  in  milk,  450 

Arnold's  steam  steriliser,  34 

Arthrogenous  spores,  138 

Ascitic  bouillon,  210 

fluid  agar    (Wassermann),   213 

Ascomycetae,  128 

Ascopores,  129 

Asparagin  Media    (Frank el    and 

Voges),  183 
(Uschinsky),  183 

Aspergillus,  127 

Atmospheric  conditions,  295 

Attenuating     the     virulence     of 
organisms,  321 

Autoclave,  37 
to  use,  37 

Automatic  pipettes,  13 

Autopsies,  396 

Autopsy,  card  index  for,  402 

BACILLI,  morphology  of,  132 
Bacillus  anthracis  in  soil,  477 

in  water,  440 

coli  in  water,  detection  of,  429 
diphtherias  in  milk,  452 
enteritidis  in  water,  437 
sporogenes  in  milk,  452 

in  water,  438 

cedematis  maligni  in  soil,  477 
tetani  in  soil,  477 

in  water,  441 
tuberculosis  in  milk,  453 

antiformin  method,  502 
typhosus  in  water,  441 
Bacteria,  anatomy  of,  134 
classification  of,  131 
grouping  of,  for  study,  410 
in  tissues,  demonstration  of ,  114 
influence   of  environment   on, 

142 

metabolic  products  of,  143 
methods  of  identification,  259 
microscopical  examination   of, 
stained,  81 

unstained,  74 
physiology  of,  136 


Bacteria,  simple  stains  for,  90 
Bacterial   emulsion,    preparation 

of,  389 

enzymes,  144,  277, 
ferments,  144 
food  stuffs,  142 
toxins,  144 

Bacteriological      analyses,      gen- 
eral considerations,  415 
examination  of  blood,  377 
Base  of  microscope,  50 
Basidium,  128 

Beer  wort,  preparation  of,  175 
Beetroot  media,  200 
Beggiotoa,  morphology  of,  133 
Benzole  bath,  256 
Berkefeld  filter,  42 
Beyrinck's  solution  I,  197 

II,  198 

Bile  salt  agar  (MacConkey),  205 
broth,  double  strength,   199 

(MacConkey),  180 
Biochemical  examination  of  cul- 
tures, 276 

Biochemistry  of  bacteria,  276 
Biological  differentiation  of  bac- 
teria, 249 

Bipolar  germination,  140 
Bismarck  brown,  94 
Blastomycetes,    morphology    of, 

129 

Blood  agar,  171,  214 
plates,  animal,  251 

human,  250 
(Washbourn),  214 
bacteriological  examination  of, 

377 

cells,  washing  of,  388 
collection    of,    for    serological 

examination,  379 
films,  preparations  of,  376 

staining  of,  97 
histological     examination     of, 

373 

pipettes,  ii 

serological  examination  of,  378 

stains,  97 

Blood-serum     (Councilman    and 
Mallory),  208 

inspissated,  168 

(LoefHer),  208 

(Lorrain  Smith),  208 
Blowpipe  table,  9 
Body  tube  of  microscope,  50 
Bohemian  flask,  4 
Boiling  water,  33 
Bone  marrow,  films,  preparation 

of,  400 

Bordet-Gengou  reaction,  393 
Boric  acid  in  milk,  test  for,  442 


INDEX 


507 


Botkin's  anaerobic  method,  243 

Bouillon,  preparation  of,  163 

Brain  extract,  149 

Bread  paste,  193 

Brilliant  green    agar    (Conradi), 

206 
bile  salt  agar  (Fawcus),  206 

Brownian  movement,  79 

Buchner's  anaerobic  method,  238 

Bulloch's  anaerobic  method,  245 
tubes  for  permanent  prepara- 
tions, 407 

Bunge's  mordant,  104 

Burri's  Chinese  ink  stain,  77 

Butter,  analysis  of,  457 
qualitative  analysis  of,  458 
quantitative  analysis  of,  457 

CADAVER,    preparation    of,    for 

'     autopsy,  397 

Cages  for  guinea-pigs,  343 

for  laboratory  animals,  341 

for  mice,  342 

for  rabbits,  343 

for  rats,  342 
Calculated   figure  for  weight  of 

medium  mass,  166,  167 
Cambier's  candle  method  of  iso- 
lating colityphoid  groups,  438 
Camera  lucida,  62 
Capaldi-Proskauer  medium,    No 

I,  186 
No  II,  187 
Capillary  pipettes,  10 

graduated,  13 
Capitate  bacilli,  139 
Capsule  formation,  134 

of  bacteria,  134 

thermo-regulator,  218 
Capsules,    collodion,    inoculation 

of,  357 

preparation  of,  357 
glass,  6 
to  clean  infected,  20 

new,  1 8 
to  stain,  99 
to  sterilise,  31 

Carbohydrate    media,     prepara- 
tion of,  177 
Carbolic  acid  as  a  germicide,  27, 

481 
method    of    isolating    coli-ty- 

phoid  group,  437 
Carbolised  agar,  202 
bouillon,  202 
gelatine,  202 
Carbon  dioxide  in  cultures,  test 

for,  289 

Card  index,  336,  402 
Carrot  media,  200 


Cedarwood     oil     for     immersion 

lens,  88 

Cell  wall  of  bacteria,  134 
Celloidin    sacs,  manufacture    of, 

Cellular  incubator,  216 
Centrifugal    machine    for    blood 

and  serum  work,  327 
for  milk  work,  447 
Centrifugalised  milk,  449 
Centrigade    degrees,    conversion 

of,  494 
Chemical    products   of   bacteria, 

China    green    agar     (Werbitski), 

207 

Chloroform   as  an  antiseptic,  27 
Chromatic  aberration,  56 
Chromogenic  bacteria,  131 
Chromoparous  bacteria,  144 
Chromophorus  bacteria,  144 
Citrated  blood  agar,  191 
Cladothrix,  morphology,  193 
Classification  of  bacteria,  131 

of  fungi,  126 
Clavate  bacilli,  139 
Clearing  films  with  acetic  acid,  82 
Clostridium,  139 
Coarse  adjustment,  51 
Cobweb  micrometer,  66 
Cocaine,  345 

Cocci,  morphology  of,  131 
Coccidium  infection,  339 
Coefficient,  inferior  lethal,  312 

of  inhibition,  311 

phenol,  489 

superior  lethal,  313 
Cohn's  solution,  191 
Cold  incubator,  217 
Coli-typhoid    group,    differential 

table,  433 
in  milk,  451 
in  soil,  477 
isolation  of,  432 
members  of,  430 

Collection  of  blood  for  bacterio- 
logical examination,  378 
for  media  making,  168 

of  milk  samples,  443 

of  pathological  material  during 
life,  373 

of  pus,  373 

of  soil  sample,  471 

of  water  samples,  416 
Collodion  capsules,  357 

sacs,  manufacture  of,  357 
Colonies  of  bacteria,  edges,  267 
Coloured  light,  action  of,  309 
Columella,  127 
Comparative  haemocytology,  374 


5o8 


INDEX 


Complement,  definition   of,  325 

fixation  test,  393 
Concentration   method  in  water, 

analysis,  434 
Condenser  achromatic,  54 

dark  ground,  60 

paraboloid,  60 

substage,  54 
Condidium,  128 
Continuous  sterilisation,  36 
Contrast  stains,  93 
Corrosive    sublimate  (Lang),  82 
Cotton- wool  filter,  40 
Counterstaining  films,  84 
Counting  plate  colonies,  423 
Cover-slip  films,  81 

to  clean  new,  22 

used,  24 

Crates  for  test-tubes,  31 
Cream,  analysis  of,  457 

qualitative  analysis  of,  458 

quantitative  analysis  of,  457 
Crenothrix  morphology,  133 
Criteria  of  infection,  370 
Criterion  of  immunity,  324 
Cultural    characters,    macroscop- 

ical  examination,  261 
Culture  flask,  Guy's,  5 
Kolle,  4 
Roux,  5 

Cuneate  bacilli,  139 
Cutaneous  inoculation,  352 

DARK  ground  condenser,  60 

illumination,  87 
Daughter  cells,  129 
Daylight,  diffuse,  action  of,  308 
Decimal  scales,  340 
Decolourising    agents,  84 
Definition  of  objective,  56 
Depilatory  powder,  346 
Description  of  plate  culture,  261 
Descriptive  terms,  261 
Desiccation,  effects  of,  306 

table,  501 

Desiccator,  Mueller's,  307 
Dextrose  solution,  preparation  of, 

178 

Diaphragm,  ins,  53 
Diastatic  enzymes,  tests  for,  278 
Differential   atmosphere  cultiva- 
tion, 257 

incubation,  255 

media,  255 

staining,  108 

sterilisation,  256 
Diluting  chamber,  248 
Dilution  by  teat  pipette,  383 

of  serum,  382 

tables,  498 


Dilutions,  preparations  of,  496 
Diphtheria,  bacillus  of,  in  milk, 

.452 

Diplobacilli,  morphology  of,  133 
Diplococci,  morphology  of,  133 
Diplococcus  pneumonias,  immuni- 
sation against,  322 
Discontinuous  sterilisation,  36 
Discs  of  plaster-of- Paris,  192 
Disinfectants,  action  of,  310 
chemical,  27 
testing  of,  480 
Dissociating   fluid,    Price  Jones, 

400 

Dosage  of  inoculum,  316 
Double  nosepiece,  58 
stains  for  spores,  106 
sugar  agar  (Russell),  207 
Drop-bottle,  73 
Dry  heat,  28 
Dunham's  solution,  177 
Dyes,  aniline,  83 

EARTHENWARE    box    for     dirty 

slides,  70 
Earthy  salts  agar   (Lip man  and 

Brown),  197 

Edge  of  individual  colonies,  char- 
acters of,  267 
Egg  albumin  agar,  213 

broth,  (Lipschuetz) ,  213 
media  (Dorset),  preparation  of, 

174 

inspissated,  212 
(Lubenan),  209 
(Tarchanoff  and  Kolesmi- 

koff),  212 
to  clear  nutrient  media  with, 

1 66 

Ehrlich's  eyepiece,  55 
Eikonometer,  65 
Eisenberg's     milk-rice     medium, 

189 

Electric  dental  engine,  360 
signal  clock,  38 
warm  stage,  59 
Elevation  of  colonies,  263 
Eisner's  gelatine,  204 

method  of  isolating  coli:    ty- 
phoid group,  438 
Endogenous  spores,  138 

varieties  of,  139 
Endo-germination,  139 
English  proof  agar,  Blaxall,   193 
Enumerating  colonies  on  plates, 

423 
discs,  Jeffer's,  424 

Pakes',  424 

Enrichment     method    in    water 
analysis,  427 


INDEX 


509 


Enumeration  of  micro-organisms, 

423 

Environmental  conditions,  142 
Enzyme    production,    investiga- 
tion of,  277 
Eosin,  93 

Equatorial  germination,  140 
Erlenmeyer  flask,  4 
Ernstschen  Koerner,  136 
Esmarch's  roll  culture,  226 

water  collecting  bottle,  417 
Estimation  of  reaction  of  media, 

280 
Ether  flame,  28 

soluble  acids,  284 
Eucaine,  345 
Exalting  virulence  of  organisms, 

320 

Examination  of  milk,  441 
Experimental    infections,    study 
of,  during  life,  370 

inoculation  of  animals,  332 
Extracellular  toxins,  144 
Eyepiece,  Ehrlich,  55 

micrometer,  63 
Eyepieces,  55 
Eye-shade,  57 

FAHRENHEIT  degrees,  conversion 

of,  495 

Feeding  experiments,  369 
Fermentation  reactions,  279 

tubes,  17 

Field  of  objective,  56 
Filar  micrometer,  66 
Filling  tubes,  etc.,  with  medium, 

1 60 

Film  preparations,  81 
fixing,  8 1 
making,  81 
mounting,  85 
staining,  83 

Filter  candle,  closed,  47 
open,  43 

testing  efficiency  of,  478 
to  disinfect,  28 
to  sterilise,  29 
flask,  6 

gapers,  to  fold,  156 
;ers,  cotton- wool,  40 

porcelain,  42 

testing  of,  478 
Filtration,  40 

by  aspiration,  42 

of  media,  156 

under  pressure,  45 
Fine  adjustment,  51 

spindle  head,  52 
Fish,  analysis  of,  460 

bouillon,  190 


Fish  gelatine,  190 

gelatine-agar,  190 
Fishing  colonies,  253 
Fission,  reproduction  by,  136 
Fixation,  81 

by  heat,  81 

of  tissues,  114 
Fixing  fluids,  for  films,  82 
Flagella,   classification  of  bacilli 
by,  136 

to  stain,  101 
Flask  Bohemian,  4 

Erlenmeyer,  4 

filter,  6 

Kitasato'a  serum,  6 

Kolle's  culture,  4 
Flasks  and  test  tubes,  to  plug,  24 

to  clean  dirty,  20 
new,  1 8 

to  sterilise,  31 
Fleischwasser,  148 
Fluid     cultures,    description    of, 
271 

media,  146 

Foot  of  microscope,  50 
Formaldehyde  in  milk,  Hehner's 

test  for,  442 
Formalin  method  of  preserving 

cultures,  407 
tissues,  404 

Fractional  sterilisation,  33 
Fraenkel  and  Voge's  solution,  183 
Fraenkel's  earth  borer,  472 
Freezing  method  for  sections,  1 1 5 
French  Mannite  Agar  (Sabour- 
aud),  193 

proof  agar  (Sabouraud),  193 
Fresh  preparations  of  bacteria,  74 
Friedlander's  capsule  stain  for 

sections,  123 

Frost's  mounting  fluid,  406 
Frozen  sections,   rapid    method, 

116 
Fuchsin,  92 

agar    (Braun),  205 

sulphite  agar    (Endo),  206 

GAS  analysis,  qualitative,  290 
quantitative,  290 

collecting  apparatus,  291 

generators,  242 

production  by  bacteria,  289 

tubes  for  media,  161 
Gasperini's  solution,  193 
Gelatin  agar,  193 

preparation  of,  164 

surface  plates,  23 1 
General  anaesthetics,  345 
Gentian  violet,  91 
German  lined  paper,  69 


INDEX 


Germicides,  27 

testing  power  of,  480 
Germination,  140 
Geryk  air-pump,  43 
Glass  apparatus  in  common  use, 

3 

to  clean,  18 
Glass-cutting  knife,  8 
Glucose  formate  agar  (Kitasato), 

180 

bouillon  (Kitasato),  180 
gelatine  (Kitasato),  180 
Glycerinated  potato,  209 
Glycerine  agar,  209 
blood-serum,  208 
bouillon,  209 
potato  bouillon,  203 

broth,  203 

Goadby's  gelatine,  214 
Gonidium,  128 
Goniodophore,  128 
Graduated  capillary  pipettes,  13 

pipettes,  6 
Gram-Claudius'  differential  stain, 

109 

Gram's  differential  stain,  108 
Gram-Weigert  for  sections,    121, 

122 

Gram-Weigert's  differentiail  stain, 

109 
modified,  no 

Grease  pencils,  72 

Grouping  of  bacteria  for  study, 
410 

Guarded  trepine,  360 

Guarniari's  agar  gelatine,  194 

Guinea-pig  cages,  343 
holder,  350 

Gulland's  solution,  82 

Gum  solution,  preparation  of ,  116 

Guy's  culture  bottle,  5 

Gypsum  blocks  (Engel  and  Han- 
sen),  192 

HJEMATIN,  95 
Haematocytometer,  248 
Haematoxilin,  95 
Haemolysin,  definition  of,  326 

preparation  of,  327 

storage  of,  331 
Hsemolytic  serum,   titration  of, 

328 
Hanging- block     culture      (Hill) , 

235 
Hanging-drop  cultures,  233 

examination  of,  86,  79 

preparation  of,  78 

permanent  staining  of,  80 

slides,  70 
Hardening  tissues,  114 


Haricot  agar,  200 

bouillon,  200 
Hay  infusion,  200 
Hearson's  water  bath,  299 
Heat   effect  of,  299 
Hehner's  test,  442 
Heiman's  serum  agar,  210 
Hesse's  anaerobic  culture  method, 

.237 
Histological  examination  of  blood, 

373 

Holder  for  guinea-pigs,  350 
Hot  air,  29 

steriliser,  30 
to  use,  31 
incubator,  217 
Hot- water  funnel,  158 
Human  blood  agar  plates,  250 
Huyghenian  eyepiece,  55 
Hydrogen,  generating  apparatus, 

242 

in  culture,  test  for,  289 
peroxide  in  milk,  test  for,  442 
Hyphomycetes,    morphology    of, 

126 
reproduction  of,   126 

ICE-BOX,  for  water  samples,  419 
Ice  cream,  analysis  of,  457 
Illuminant  for  microscope,  67 
Immune  body,  393 
Immunisation,  methods  of,  321 
Imperial  system,  492 

factors   for   converting,    493 
Impression  films,  85 
Incubators,  216 
Index  cards,  336,  403 
Indol,  test  for,  286 
Infection,  definition  of,  370 

general     observations     during 
life,  371 

results  of,  404 
Influence     of     environment     on 

bacterial  growth,  142 
Inhalation,  fluid  inoculum,  365 

powdered  inoculum,  366 
Inhibition  coefficient,  310,  311 
Inoculation  card  index,  336 

cutaneous,  352 

intracranial,  360 

intramuscular,  355 

intraocular,  362 

intraperitoneal,  355 

intrapulmonary,  363 

intravenous,  363 

of  collodion  capsules,  357 

subcutaneous,  353 

syringe,  344 
Inoculum,  character  of,  346 

preparation  of,  346 


INDEX 


Inosite-free  media — bouillon(Dur- 

ham),  183 

Inseparate  toxins,  144 
Intermittent  sterilisation,  36 
Intracellular  toxins,  144 
Intracerebral  inoculation,  362 
Intracranial  Inoculation,  360 
Intragastric    inoculation,     large 

animals,  367 
Marks  method,  367 
Intramuscular  inoculation,  355 
Intraocular  inoculation,  362 
Intraperitoneal  inoculation,  355 
Intrapulmonary  inoculation,  363 
Intravenous  inoculation,  363 
In  vacuo  anaerobia  cultures,  289 
Invertin  enzymes,  tests  for,  279 
Involution  forms,  137 
Iodine  solution,  108 
Iron  bouillon,  185 

peptone  solution  (Pakes),    185 
Isolation  by  animal  experiments, 

258 

by  differential  atmosphere  ,257 
incubation,  255 
media,  255 
sterilisation,  256 
by  dilution,  248 
by  plate  cultures,  250 
subcultures,    preparatioin    of, 
254 

JEFFER'S  counting  disc,  424 

Jenner's  stain,  97 

Jores'  mounting  fluid,  405 

KAISERLING  fixing  solution,  405 
Kanthack's  serum  agar,  211 
Killed  cultivations,  318 
Kipp's  hydrogen  apparatus,  242 
Kitasato's  serum  flask,  6 
Klebs-Loeffler   bacillus   in   milk, 

452 

Koch's  steam  steriliser,  34 
Kohle's  culture  flask,  4 

LAB  enzymes,  test  for,  279 
Laboratory  animals,  335 

comparative     haematocytol- 

ogy  of,  374 

normal  temperature,  372 
regulations,  I 

Lactose  litmus  agar  (Wurtz),  203 
bouillon,  203 
gelatine  (Wurtz),  203 
Lakmus  Molke,  203 
Lang's  solution,  82 
Lead  bouillon,  185 
peptone  solution,  186 


Leishman's  stain,  98 
for  sections,  1 25 
Lemco  broth,  163 
Leptothrix,  morphology,  133 
Lethal  dose,  minimal,  316 
Leviditi's  staining  method,  124 
Light,  action  of,  308 
Liquefiable  media,  147 
Liquid  soap,  346 
Lithium  carmine,  96 
Litmus  bouillon,  186 
gelatine,  202 
milk  cultures,  description  of, 272 

preparation  of,  172 
nutrose   agar    (Drigalski-Con- 

radi),  205 
whey,  195 
agar,  196 
gelatine,  196 
(Petruschky),  195 
Local  anaesthetics,  345 

reaction  to  infection,  372 
Locomotive  movement,  80 
Lceffler's  capsule  stain,   103 

serum,   208 

Lophotrichous  bacilli,  136 
Lorrain     Smith     electric     warm 

stage,  59 
serum,  208 

Lugol's  solution,  to  prepare,   108 
Lysol,  27 

MACCONKEY'S  capsule  stain,  99 

media,  180,  199,  205 
MacCrorrie's  capsule  stain,  103 
Macroscopical     examination     of 

cultures,  261 
Malachite  green  agar    (Lceffler), 

207 
Malt  extract  solution  (Herschell) , 

196 

Margin  of  individual  colonies,  267 
Martin's  filtering  apparatus,  320 
Material  for  inoculation,  346 
Mayer's  albumin,  120 
Mean  phenol  coefficient,  490 
Measuring  bacteria,  61 
Meat,  bacteriological  analysis  of, 

460 
extract  preparation  of,  148 

reaction  of,  149 

Mechanical    separation    of    bac- 
teria, 249 
stage,  52 

Media,  filtration  of,  156 
preparation  of,  163 
aerobic  culture,  222 
aesculin  agar,  204 
agar- agar,  167 
agar  gelatine  (Guarniari),  194 


INDEX 


Media,  preparation  of  anaerobic 

culture,  1 80 

animal -tissue  (Frugoni),  210 
ascitic  bouillon,.  210 

fluid  agar   (Wassermann), 

213 
asparagin      (Fraenkel-     and 

Voge's),  183 
(Uschinsky),  183 
beer  wort,  175 
beetroot,  200 
Beyrinck's  solution  I,   197 

II,  198 
bile  salt  agar  (MacConkey), 

205 

broth  (MacConkey),  180 
double  strength,  199 
blood  agar  (Washbourn),  214 
blood-serum,  168 

(Councilman     and     Mai- 
lory),  208 
(Loeffler),  208 
(Lorrain  Smith),  208 
bouillon,  163 
bread  paste,  193 
brilliant    green    agar    (Con- 

radi),  206 
bile  salt  agar  (Fawcus), 

206 
Capaldi-Proskauer,  No.  1, 186 

No.  II,  187 
carbohydrate,  177 
carbolised  agar,  202 
bouillon,  202 
gelatine,  202 
carrot,  200 
China   green   agar    (Werbit- 

ski),  207 

titrated  blood  agar,  171 
Cohn's  solution,  191 
dextrose  solution,  178 
double  sugar  agar  (Russell), 

207 
earthy  salt  agar  (Lip  man  and 

Brown),  197 
egg  Dorset,  174 
Lubenau,  209 

egg-albumen,  inspissated,  212 
(Tarchanoff     and    Koles- 

nikoff),  212 
egg-albumin  agar,  213 

broth  (Lipschuetz),  213 
English  proof  agar  (Blaxall), 

193 

fish  bouillon,  190 
gelatine,  190 

agar,  190 
fluid,  146 

French   mannite  agar   (Sab- 
ouraud),  193 


Media,   preparation     of    French 

proof  agar(Sabouraud),  193 

Fuchsin   agar    (Braun),    205 

sulphite  agar  (Endo),   206 

gelatine,  193 

agar,  193 
glucose  formate  agar   (Kit- 

asato),  1 80 

bouillon  (Kitasato),  180 
gelatine  (Kitasato),  180 
glycerinated  broth,  209 

potato,  209 
glycerine  agar,  209 
blood-serum,  208,  209 
bouillon,  209 
potato  bouillon,  203 
gypsum  blocks   (Engel  and 

Hansen),  192 
haricot  agar,  200 

bouillon,  200 
hay  infusion,  200 
inosite    free-bouillon    (Dur- 
ham), 183 
iron  bouillon,  185 

peptone  solution  (Pakes), 

185. 

lactose  litmus  agar  (Wurtz), 
203 

bouillon,  203 
gelatine  (Wurtz),  203 
lakmus  molke,  203 
lead  bouillon,  185 

peptone  solution,  186 
lemco  broth,  163 
liquefiable,  147 
litmus  bouillon,  186 
gelatine,  202 
milk,  172 
nutrose    agar    (Drigalski- 

Conradi),  205 
whey,  195 
agar,  196 
gelatine,  196 
(Petruschky),  195 
malachite  green  agar  (Loeff- 
ler), 207 
malt  extract  solution  (Hers- 

chell),  196 
milk,  172 

rice  (Eisenberg),  189 

(Soyka),  189 
Naegeli's  solution,  191 
Naehrstoff  agar  (Hesse  and 

Niedner),  199 

neutral  litmus  solution,   179 
nitrate  bouillon,  185 
peptone  solution  (Pakes), 

1 86 

nutrient,  146 
agar- agar,  167 


INDEX 


513 


Media,    preparation   of  nutrient 
bouillon,  163 

gelatine,  164 
nutrose  agar  (Eyre),  172 
oleicacid  agar  (Fleming),  201 
Omeliansky's  nutrient  fluid, 

189 

Parietti's  bouillon,  202 
parsnip,  200 
Pasteur's  solution,  191 
peptone  rosolic  acid    water, 

1 86 

water  (Dunham),  177 
plaster-of-Paris  discs,  192 
potato,  174 

gelatine  (Eisner),  204 

(Goadby),  214 
proteid  free  broth    (Uschin- 

sky),  183 
rosolic  acid  peptone  solutions 

1 86 
serum,  bouillon,  210 

dextrose  water,  (Hiss),  188 

sugar,  (Hiss),  188 

water,  170 
serum-agar  (Heiman),  210 

(Kant hack  and  Stevens), 

211 

(Lib man),  212 

(Wertheimer),  211 
silicate  jelly  (Winogradsky), 

198 

solid,  147 
special,  182 
stock  nutrient,  163 
sugar,  177 

agar,  185 

(dextrose)  bouillon,  184 

gelatine,  184 
sulphindigotate  agar,  181 

bouillon  (Weyl),  181 

gelatine  (Weyl),  181 
tissue   (Noguchi),  214 
turnip,  200 
urine  agar,  188 

bouillon,  187 

gelatine,  187 

(Heller),  188 
wheat  bouillon  (Gasperini), 

193 

whey  agar,  195 
gelatine,  195 
wine  must,  192 
Winogradsky 's  solution  (for 
nitric  organisms),  198 
(for  nitrous  organisms), 

198 

wood  ash  agar,  201 
wort  agar,  176 
gelatine,  176 
33 


Media,     preparation      of    yeast 
water  (Pasteur),  191 

standardisation  of,  154 

storage  of,  in  bulk,  159 

storing  tubes  of,  161 

sore  boxes,  162 

titration  of,  150 

tubing  of  nutrient,  160 
Merismopedia,     morphology    of, 

132 
Mesophilic  bacteria,  143 

pathogenic  effects,  315 
Metabolic  end-products,  145 
Metrachromatic  granules,  136 
Metal  instruments,  to  sterilise,  28 
Metatrophic  bacteria,  131 
Methods  of  cultivation,  221 

of  identification  of  bacteria,  259 

of  inoculation,  352 

of  isolation,  248 
-    of  sterilisation,  26 
Methylene-blue,  90 
Metric  system,  492 

factors  for  converting,  493 
Meyer's  carmine,  96 
Microbes  of  indication,  426 
Micrococci,  morphology,  132 
Micrococcus,  melitensis  in  milk, 

456 
Micrometer,  filar,  66 

net,  63 

ocular,  63 

stage,  62 

Micrometry,  methods  of,  61 
Micron,  61 
Microscope,  49 
Microscopical     examination      of 

bacteria,  86 
stained,  88 
unstained,  86 

observations  of  cultures,  272 
Milk,  analysis  of,  qualitative,  446 
quantitative,  444 

condensed,  analysis  of,  444 

media,  193 

preparation  of,  172 

rice  (Eisenberg),  193 
(Soyka),  189 

samples,  collection  of,  443 

sedimenting  tubes,  449 
Minimal  lethal  dose,  316 
Mirror  for  microscope,  55 
Moeller's  stain  for  spores,  107 
Moist  heat,  32 
Molecular  movement,  79 
Monotrichous  bacilli,  136 
Motility,  examination  for,  79 

true,  80 
Moulds,  examination  of,  126 

for  paraffin  imbedding,  117,  119 


INDEX 


Mounting  film  preparations,  85 

paraffin  sections,  119 
Mouse  cages,  342 

holder,  351 

scales,  341 
Mucor  mucedo,  126 
Mucorinae,  126 
Mueller's  desiccator,  307 
Muffle  furnace,  28 
Muirs's  capsule  stain,  100 

flagella  stain,  101 
Museum  preparations  of  bacteria, 

407 

of  tissues,  404 
sealing  of,  406 
Mycelium,  126 
Mycoprotein,  135 

NAEGELI'S  solution,  191 
Naehrstoff  agar  (Hesse  and  Nied- 

ner),  199 
Naked  flame,  28 
Neisser's  stain  modified,  1 1 1 
Net  micrometer,  63 
Neutral  litmus  solution,  prepara- 
tion of,  179 

red,  94 
Nitrate  bouillon,  185 

peptone  solution  (Pakes),    186 
Nitric  organisms  in  soil,  478 
Nitroso-indol  reaction,  287 
Nitrous  organisms  in  soil,  477 
Normal  averages  (t.p.r.),  372 

serum,  375 
Nosepiece,  57 

double,  58 

triple,  58 
Novy's  anaerobic  method,  244 

jars,  245 

Nuclei,  to  stain,  105 
Nucleus  of  bacteria,  135 
Numerical  aperature,  56 
Nutrient  media,  146 
Nutrose  agar  (Eyre),  preparation 

of,  172 

OBJECT  marker,  61 

Objectives,  55 

Oblique  tube  cultures,  223 

Ocular  micrometer,  63 

Oculars,  55 

Oese,  platinum,  71 

Oidium,  128 

Oil  of  garlic,  27 

of  mustard,  27 

Oleic  acid  agar  (Fleming),  201 
Omeliansky's   nutrient  fluid,  189 
Operation  tables  (Eyre's),  352 

(Tatin's),  351 
Opsonic  index,  393 


Opsonic  index,  determination  of, 

390 

Opsonin,  387 
Optical  characters     of     colonies, 

267 
Optimum     reaction  of  medium, 

determination  of,   305 
temperature,  determination  of, 

298 

Organisms  of  suppuration,  409 
Orsat-Lunge  gas  apparatus,  292 
Orth's  carmine,  96 
Oxford  stain  for  Actinomyces,  1 12 
Oysters,  analysis  of,  463 

Pake's  counting  disc,  424 

filter  reservoir,  45 
Papier  chardin,  158 
Pappenheim's  stain,  HI 
Paraboloid  condenser,  60 
Parachromophorous  bacteria,  144 
Paraffin  method  for  sections,    117 

sections,  mounting  of,  119 

to  stain,  121 

Paratrophic  bacteria,  131 
Parietti's  bouillon,  202 

method     of    isolating    coli-ty- 

phoid  group,  437 
Parsnip  medium,  200 
Passages  of  virus,  320 
Pasteur-Chamberland  filter,  42 
Pasteur's  pipettes,  10 

solution,  191 
Pathogenesis,    investigation    of, 

315 

Pathogenic  bacteria,  131 
study  of,  408 

Pediococci,  morphology  of,  132 

Penicillium,  128 

Peptone  rosolic  acid  water,  186 
water  (Dunham),   preparation 
of,  177 

Percentage  formula,  496 

Perchloride  of  mercury,  27 

Perisporacse,  127 

Peritrichous  bacilli,  136 

Permanent    preparations  of  bac- 
teria, 407 

of  tissues,  404 

Petri's  dishes,  6 

Phagocytic  index,  392 

Phenol  coefficient,  489 
production,  test  for,  287 

Photogenic  bacteria,  131,  144 

Physiological  filter,  156 

Picric  acid  solution,  121 
(Spengler's),  112 

Picrocarmine,  97 

Pigment  production,  observations 
on,  288 


INDEX 


515 


Pipettes,  automatic,  13 
blood,  ii 
capillary,  10 
cases  for,  7 
graduated,  6 

capillary,  13 
Pasteur's,  10 
sedimentation,  16 
standard  graduated,  7 
teat,  10 
throttle,  13 
to  clean  infected,  20 

new,  1 8 
to  sterilise,  31 
Piridin  method  of  staining  spiro- 

chaetes,  124 

Pi tfi eld's    flagella   stain,  103 
Plasmolysis,  135 
Plaster- of -Paris  discs,  192 
Plate  box,  7 

cultures,  description  of,  261 

preparation  of,  226 
levelling  stand,  228 
Plates,  Petri's,  6 

to  clean  infected,  20 

new,  1 8 
to  sterilise,  31 
Platinum  needles,  71 

method  of  mounting,  71 
Pleomorphism,  133 
Polar  germination,  140 

granules,  136 
Polkoerner,  136 
Polychrome  blood  stains,  97 
Pooled  serum,  379 
Porcelain  filter,  42 
Berkefeld,  42 
Chamberland,  42 
Doulton,  42 

Post-mortem  examination  of  ex- 
perimental animals,  396 
Potato  gelatine  (Eisner),  204 

(Goadby),  214 
medium,  preparation  of,  174 
Potted  meat,  analysis  of,  460 
Pouring  plates,  227 
Preparation  of  experimental  ani- 
mals, 335 

Preservatives  in  milk,  442 
Pressure  temperature  table,    500 
Primary  colours,  action  of,  309 
Proteid  free  broth    (Uschinsky), 

183 

Proteolytic    enzymes,    tests    for, 

277 

Prototrophic  bacteria,  131 
Psychrophilic  bacteria,  143 

pathogenic  effects,  315 
Pus,  collection  of,  373 
Pyrogallic  acid  solution,  293 


QUALITATIVE  analysis  of  air,  470 

of  milk,  446 

of  sewage,  467 

of  soil,  476 

of  unsound  meat,  462 

of  water,  426 
Quantitative  analysis  of  air,  468 

of  milk,  444 

of  sewage,  466 

of  soil,  473 

of  unsound  meat,  460 

RABBIT  cages,  343 

scabies,  treatment  of,  338 
scales,  340 

Raising  virulence  of    organisms, 
320 

Ramsden's  micrometer,  66 

Range  of  medium  reaction,  meas- 
urement of,  305 
of  temperature,    measurement 
of,  298 

Rat  cages,  342 

Raw  milk,  Saul's  test  for,  442 

Reaction  of  medium,  305 
optium,  305 
range  of,  305 
scale,  153 

Reduced  pressure  and  tempera- 
ture table,  501 

Reducing  agents,  production,  389 
tests  for,  289 

Reduction  of  nitrates,  389 

Reichert's  thermo- regulator,   218 

Relation  of  bacteria  to  environ- 
ment, 142 

Removal  of  material  from  culture 
tubes,  74 

Rennin  enzymes,  tests  for,  279 

Reproduction  of  bacteria,  136 

Resistance  glass,  6 
to  lethal  agents,  306 

Resting  stage  of  bacteria,  137 

Restrictions    upon    experimental 
inoculations,  334 

Ribbert's  capsule  stain,  101 

Roll  cultures,  226 

Rosplic  acid  peptone  solution,  186 

Rosindol  reaction,  286 

Roux's  anaerobic  culture  method, 

237 

culture  bottle,  5 

SABOURAUD'S  medium,  193 
Saccharomyces,    morphology  of, 

1-29 

Safranine,  94 

Salicylic  acid  in  milk,  test  for,  443 
Saprogenic  bacteria,  131 
Sarcinse,  morphology  of,  132 


INDEX 


Saul's  test,  442 
Scales,  decimal,  340 

trip,  164 

Scalpels,  to  sterilise,  32,  33 
Schallibaum's  solution,  121 
Scheme  for  study  of  bacteria,  259 
Schizomycetes,   classification    of, 

I3i 

morphology  of,  131 
Scissors,  to  sterilise,  32 
Sealing  museum  jars,  406 
Searing  iron,  397 
Sections,  special  staining  methods 

for,  121 
Sedimentation  pipettes,  16 

tubes,  9 

Selecting  objectives,  57 
Sensitising  red  blood  cells,  395 
Serial  cultivations,  251 
Serological  examination  of  blood, 

378 
Serum  agar  (Heiman),  210 

(Kanthack     and     Stevens), 

211 

(Libman),  212 
plates,  250 
(Wertheimer),  21 1 
bouillon,  210 
collection  of,  379 
dextrose  water  (Hiss),  188 
inspissator,  169 
sugar  media  (Hiss),  188 
water,  preparation  of,  170 
Sewage,  analysis  of,  qualitative, 

467 

quantitative,  466 
Shake  cultivations,  225 
description  of,  271 
Shape  of  colonies,  262 
Shaving    experimental    animals, 

349 

Shellfish,  analysis  of,  463 
Silicate  jelly  (Winogradsky),    198 
Single  stain  for  spores,  106 
Size  of  colonies,  262 
Slanted  tube  cultures,  223 
Slides,  to  clean  new,  22 

used,  23 
Smear  culture,  224 

description  of,  268 
Soap  liquid,  346 
Soda  solution,  storage  of    stock, 

154 
Sodium  bicarbonate  in  milk,  test 

for,  443 

Soil,  analysis  of,  qualitative,  476 
quantitative,  473 
collection  of  samples,  471 
Solid  media,  147 
Soluble  toxins,  144 


Soyka's  milk  rice,  189 
Spear-headed  spatula,  402 
Special  media,  182 
Specific  serum,  379 
dilution  of,  382 
Spherical  aberration,  55 
Spirillum,  morphology  of,  133 
Spirochasta,  morphology  of,    133 
Spirochaetes  in  tissues,  to   stain, 

124 

Spleen  extract,  149 
Sporangium,  127 
Spore    formation,    arthrogenous, 

138 

endogenous,  138 
method  of,  138,  273 

germination,  method  of,    140, 

274 

observation     of,     140,     273 
Spores,  characters  of,  139 

classification  of,  139 

double  stain  for,  106 

to  stain,  106 
Stab  culture,  224 

description  of,  265 
Stage  micrometer,  62 

of  microscope,  52 
Staining  methods,  90 

paraffin  sections,  121 

reactions  of  bacteria,  274 
Stains  intra-vitam,  77 

negative  (Burri),  77 

rack  for,  72 
Standard  graduated  pipettes,  7 

soda  solution,  154 
Standardisation  of  media,  154 
Standardising  bouillon,  155 
Staphylococci,   morphology,    132 
Staphylococcus  in  milk,  456 
Steam  steriliser,  Arnold,  35 
Koch,  35 
to  use,  35 

streaming,  35 
Sterigma,  127 
Sterilisation  by  chemicals,  27 

by  dry  heat,  28 

by  filters,  40 

by  moist  heat,  32 

by  streaming  steam,  35 

by  superheated  steam,  36 

of  albuminous  liquids,  32 

of  gases,  40 
Sterilising  agents,  26 
Stichcultur,  224 
Stock  dilutions,  497 

nutrient  media,  163 

plate  for  isolation  work,  253 
Storage  of  media  in  bulk,  159 

of  tubed  media,  161 
Store  boxes  for  media,  161 


INDEX 


517 


Streak  culture,  224 

description  of,  268 
Streaming  movement,  80 

steam,  35 

Streptobacilli,  morphology,  133 
Streptococci  in  soil,  477 

in  water,  detection  of,  432 

morphology  of,  132 
Streptococcus  pyogenes  longus  in 

milk,  455 

Streptothrix,  morphology  of,  133 
Strichcultur,  223 
Structure,  internal,    of    colonies, 

265 

Study  of  pathogenic  bacteria,  408 
Subcutaneous  inoculation,  353 
Subdural  inoculation,  361 
Substage  condenser,  54 
Sugar  agar,  185 

dextrose  bouillon,  184 

gelatine,  184 

media,  preparation  of,  177 
Sulphindigotate  agar,  181 

bouillon  (Weyl),  181 

gelatine  (Weyl),  181 
Sulphuretted    hydrogen   in    cul- 
tures, test  for,  290 
Sun-light,  action  of,  309 
Superheated  steam,  36 
Superior  lethal  coefficient,    310, 

„  3I3 

Suppuration,  organisms  of,  409 

Surface  characters  of  colonies,  264 

plates,  230 

Surgical  motor,  electric,  360 
Swarm  spores,  127 
Syringe  for  subcutaneous  inocu- 
lation of  solid  material,  354 

hypodermic,  344 

TATIN'S  operating  table,  351 
Taxonomy,  262 
Teat-^pipettes,  10 
Temperature,  action  of,  299 

optimum,  298 

pressure  table,  500 

range,  298 

taking,  340 

Test  objects  for  objectives,  57 
Testing  niters,  478 
Test-tubes,  3 

to  clean  infected,  19 
new,  1 8 

to  plug,  24 

to  sterilise,  31 

Tetracocci,  morphology  of,  132 
Thermal  death-point,  143 
determination  of,  298 
of  spores,  301,  304 
of  vegetative  forms,  298,  303 


Thermophilic  bacteria,  143 
Thermo-regulators,         Hearson's 

capsule,  218 
Reichert's,  218 
Thionine  blue,  92 
Thiothrix,  morphology  of,  133 
Thresh's  water  collecting  bottle, 

418 

Throttle  pipettes,  13 
Tinned  meat,  analysis  of,  460 
Tissue  medium  (Noguchi),  214 

stains,  95 
Tissues  for  sectioning,  fixing,  114 

freezing,  116 

hardening,  114 

imbedding,  118 

preparation  of,  114 

washing,  115 
Titration  of  media,  150 
Tortulae,  differentiation  from  sac- 

charomyces,  130 
Total  acidity,  280 
Toxins,  testing  of,  318 
Trephines,  360 
Triple  nosepiece,  58 
True  motility,  80 
Tube  cultures,  preparation  of,  222 

length,  50  > 
Tubercle  bacillus  in  milk,  453 

to  stain,  up,  124 
Tuberculous  guinea-pig,  cadaver 

of,  454 

Tubing  nutrient  media  ,160 
Turnip  media,  200 

UNNA-PAPPENHEIM'S    stain     for 

sections,  123 

Unsound  meat,  analysis  of,  460 
Urine  agar,  188 

gelatine,  187 
(Heller),  188 

media  bouillon,  187 
Uschinsky's  solution,  183 

VALENCY  of  specific  sera,  386 
Van  Ermengem's  flagella  stain, 

104 

Vegetative  stage  of  bacteria,  136 
Vesuvin,  94 

Vibrio  choleras  in  milk,  452 
in  water,  439 

morphology  of,  133 
Virulence,  attenuating,  321 

of  organisms,  320 

raising,  320 

Vivisection  license,  334 
Voges  holder,  350 
Volatile  oils  as  disinfectants,  27 

WARM  stage,  58 


INDEX 


Washing  red  blood  cells,  388 

tissues,  115 

Water,  analysis'of  .qualitative,  426 
quantitative,  416 

steriliser,  33 
Weighing  animals,  340 
Welch's  capsule  stain,  101 
Wertheimer's  serum  agar,  211 
Wheat  bouillon  (Gasperini),  193 
Whey  agar,  195 

gelatine,  195 
Wine  must,  192 
Winogradsky's  solution  I,  198 
II,  198 


Wire  crates  for  test-tubes,  3 1 
Wood  ash  agar,  201 
Working  up  plates,  252 
Wort  agar,  176 
gelatine,  176 
Wright's  anaerobic  method,  239 

YEAST  water  (Pasteur),  191 

ZIEHL-NEELSEN'S  stain,  no 
Zooglcea,  134 
Zymogenic  bacteria,  131 


SAUNDERS'  BOOKS 

OH      

Practice,  Pharmacy, 
Materia  Medica,  Thera- 
peutics, Pharmacology, 
and  the  Allied  Sciences 

W.  B.  SAUNDERS   COMPANY 

WEST  WASHINGTON  SQUARE  PHILADELPHIA 

9,  HENRIETTA  STREET,  COVENT  GARDEN,  LONDON 

«*a.^_^^_^^^.^^M.l^_^^.»-i^^^_^^— •».^^^^^^^^^_^^H^^^^^M.^^ 

Garrison's 
History  of  Medicine 

History  of  Medicine.  With  Medical  Chronology,  Bibliographic 
Data,  and  Test  Questions.  By  FIELDING  H.  GARRISON,  M.  D.,  Prin- 
cipal Assistant  Librarian,  Surgeon-General's  Office,  Washington,  D.  C. 
Cloth,  $6.00  net;  Half  Morocco,  $7.50  net. 

REPRINTED  IN  THREE  MONTHS— THE  BAEDEKER  OF  MEDICAL  HISTORY 

The  work  begins  with  ancient  and  primitive  medicine,  and  carries  you  in  a 
most  interesting  and  instructive  way  on  through  Egyptian  medicine,  Sumerian 
and  Oriental  medicine,  Greek  medicine,  the  Byzantine  period  ;  the  Mohammedan 
and  Jewish  periods,  the  Medieval  period,  the  period  of  the  Renaissance,  the  Re- 
vival of  learning  and  the  Reformation  ;  the  Seventeenth  Century  (the  age  of  indi- 
vidual scientific  endeavor),  the  Eighteenth  Century  (the  age  of  theories  and 
systems),  the  Nineteenth  Century  (the  beginning  of  organized  advancement  of 
science),  the  Twentieth  Century  (the  beginning  of  organized  preventive  medicine). 
You  get  all  the  important  facts  in  medical  history  ;  a  biographic  dictionary  of  the 
makers  of  medical  history,  arranged  alphabetically  ;  an  album  of medical  portraits  ; 
a  complete  medical  chronology  (data  on  diseases,  drugs,  operations,  etc. ) ;  a  brief 
survey  of  the  social  and  cultural  phases  of  each  period. 


SAUNDERS'    BOOKS   ON 


Musser  and  Kelly  on 
Treatment 

A  Handbook  of  Practical  Treatment.  By  82  eminent  specialists. 
Edited  by  JOHN  H.  MUSSER,  M.  D.,  and  A.  O.  J.  KELLY,  M.  D.,  Univer- 
sity of  Pennsylvania.  Three  octavos  of  950  pages  each,  illustrated. 
Per  volume:  Cloth,  $6.00  net;  Half  Morocco,  $7.50  net.  Subscrip- 
tion. 

IN  THREE  VOLUMES 
A  PRACTICE  FOR  QUICK  REFERENCE  AND  DAILY  USE 

Every  chapter  in  this  work  was  written  by  a  specialist  of  unquestioned  authority. 
Not  only  is  drug  therapy  given  but  also  dietotherapy,  serumtherapy,  organo- 
therapy, rest-cure,  exercise  and  massage,  hydrotherapy,  climatology,  electro- 
therapy, .r-ray,  and  radial  activity  are  fully,  clearly,  and  definitely  discussed. 
Those  measures  partaking  of  a  surgical  nature  have  been  presented  by  surgeons. 

The  Medical  Record 

"  The  most  modern  and  advanced  views  are  presented.  It  is  difficult  to  pick  out  any  one 
topic  that  deserves  special  commendation,  all  parts  fully  covering  their  particular  field,  and 
written  with  that  fulness  of  detail  demanded  by  the  every-day  needs  of  the  practitioner." 


Thomson's  Clinical  Medicine 

Clinical  Medicine.     By  WILLIAM  HANNA  THOMSON,  M.  D.,  LL.  D., 

formerly  Professor  of  the  Practice  of  Medicine  and  of  Diseases  of  the 
Nervous  System,  New  York   University  Medical  College.     Octavo  of 

675  pages. 

JUST  READY 

This  new  work  represents  over  a  half  century  of  active  practice  and  teach- 
ing. It  deals  with  bedside  medicine — the  application  of  medical  knowledge  for 
the  relief  of  the  sick.  First  the  meaning  of  common  and  important  symptoms  is 
stated  definitely ;  then  follows  a  chapter  on  the  use  of  remedies  and  a  classifi- 
cation of  them  ;  next  the  section  on  infections,  and  last  a  section  on  diseases  of  par- 
ticular organs  and  tissues.  It  is  medical  knowledge  applied — from  cover  to  cover. 
An  important  chapter  is  that  on  the  mechanism  of  surface  chill  and  "catching 
cold,"  going  very  clearly  into  the  etiologic  factors,  and  outlining  the  treatment. 
The  chapter  on  remedies  takes  up  non-medicinal  and  medicinal  remedies  and 
"vaccine  and  serum  therapy.  In  the  chapter  on  the  ductless  glands  the  subject  of 
internal  secretions  is  very  clearly  presented,  giving  you  the  latest  advances.  The 
infectious  diseases  are  taken  up  in  Part  II,  while  Part  III  deals  with  diseases  of 
special  organs  or  tissues,  every  disease  being  fully  presented  from  the  clinical 
side.  Treatment,  naturally,  is  very  full. 


DIAGNOSIS  AND    TREATMENT. 


Cabot's 
Differential  Diagnosis 

Differential  Diagnosis.  Presented  through  an  Analysis  of  385 
Cases.  By  RICHARD  C.  CABOT,  M.  D.,  Assistant  Professor  of  Clinical 
Medicine,  Harvard  Medical  School,  Boston.  Octavo  of  764  pages, 
illustrated  Cloth,  $5.50  net. 

THE  NEW  (2d)  EDITION 
EIGHT  LARGE  PRINTINGS 

Dr.  Cabot' s  work  takes  up  diagnosis  from  the  point  of  view  of  the  presenting 
symptom — the  symptom  in  any  disease  which  holds  the  foreground  in  the  clinical 
picture  :  the  principal  complaint.  It  groups  diseases  under  these  symptoms,  and 
points  the  way  to  proper  reasoning  in  coming  to  a  correct  diagnosis.  It  works 
backward  from  each  leading  symptom  to  the  actual  organic  cause  of  the  symptom. 
This  the  author  does  by  means  of  case-teaching. 

Chas.  Lyman  Greene,  M.D.,  University  of  Minnesota. 

"  It  is  one  of  the  most  valuable  books  that  has  been  published  in  recent  years,  or  indeed  at 
any  time." 


Morrow's  Diagnostic  and 
Therapeutic  Technic 

Diagnostic  and  Therapeutic  Technic.     By  ALBERT   S.   MORROW, 
M.  D.,  Adjunct  Professor  of  Surgery,  New  York  Polyclinic.     Octavo 
of  775  pages,  with  815  original  line  drawings.     Cloth,  $5.00  net 
JUST  THE  WORK  FOR  PRACTITIONERS 

Dr.  Morrow's  new  work  is  decidedly  a  work  for  you— the  physician  engaged 
in  general  practice.  It  is  a  work  you  need  because  it  tells  you  just  how  to  perform 
those  procedures  required  of  you  every  day,  and  it  tells  you  and  shows  you  by 
clear,  new  line-drawings,  in  a  way  never  before  approached.  It  is  not  a  book  on 
drug  therapy  ;  it  deals  alone  with  physical  or  mechanical  diagnostic  and  thera- 
peutic measures.  The  information  it  gives  is  such  as  you  need  to  know  every 
day— transfusion  and  infusion,  hypodermic  medication,  Bier's  hyperemia,  explora- 
tory punctures,  aspirations,  anesthesia,  etc.  Then  follow  descriptions  of  those 
measures  employed  in  the  diagnosis  and  treatment  of  diseases  of  special  regions  or 
organs:  proctoclysis,  cystoscopy,  etc. 
Journal  American  Medical  Association 

"The  procedures  described  are  those  which   practitioners  may  at  some  time 
on  to  perform.'* 


4  SAUNDERS'  BOOKS  ON 

Faught's  Blood-Pressure 

Blood  -  Pressure  from  the  Clinical  Standpoint.  By  FRANCIS  A. 
FAUGHT,  M.  D.,  formerly  Director  of  the  Laboratory  of  Clinical  Medi- 
cine of  the  Medico-Chirurgical  College  of  Philadelphia.  Octavo  of  281 
pages,  illustrated.  Cloth,  $3.00  net. 

THREE  PRINTINGS  IN  SIX  MONTHS 

Dr.  Faught's  book  is  designed  for  practical  help  at  the  bedside.  It  meets  the 
urgent  needs  of  the  general  practitioner,  who  heretofore  had  no  book  to  which  to 
turn  in  case  of  emergency.  Every  effort  has  been  made  to  provide  here  a  practical 
guide,  full  of  information  of  a  clinical  nature,  and  presented  in  a  way  readily 
available  for  daily  use  by  the  busy  man.  Besides  the  actual  technic  of  using  the 
sphygmomanometer  in  diagnosing  disease,  Dr.  Faught  has  included  a  brief 
general  discussion  of  the  process  of  circulation.  The  wonderful  strides  made  in 
our  knowledge  of  blood-pressure,  and  the  practical  application  of  sphygmomano- 
metric  findings  within  recent  years,  make  it  imperative  for  every  medical  man  to 
have  close  at  hand  an  up-to-date  work  on  this  subject. 


Anders  &  Boston's  Medical  Diagnosis 


A  Text-Book  of  Medical  Diagnosis. — By  JAMES  M.  ANDERS,  M.D., 
PH.D.,  LL.D.,  Professor  of  the  Theory  and  Practice  of  Medicine  and  of 
Clinical  Medicine,  and  L.  NAPOLEON  BOSTON,  M.D.,  Adjunct  Professor 
of  Medicine,  Medico-Chirurgical  College,  Philadelphia.  Octavo  of  1175 
pages,  with  443  illustrations,  a  number  in  colors.  Cloth,  $6.00  net; 
Half  Morocco,  $7.50  net. 

THE    MODERN  DIAGNOSIS 

This  new  work  is  designed  expressly  for  the  general  practitioner.  The 
methods  given  are  practical  and  especially  adapted  for  quick  reference.  The 
diagnostic  methods  are  presented  in  a  forceful,  definite  way  by  men  who  have 
had  wide  experience  at  the  bedside  and  in  the  clinical  laboratory. 

The  Medical  Record 

The  association  in  its  authorship  of  a  celebrated  clinician  and  a  well-known  laboratory 
worker  is  most  fortunate.     It  must  long  occupy  a  pre-eminent  position." 


PRACTICE   OF  MEDICINE 


Ward's  Bedside  Hematolog'y 

Bedside  Hematology.  By  GORDON  R.  WARD,  M.D.,  Fellow  oi  the 
Royal  Society  of  Medicine,  London,  England.  Octavo  of  394  pages, 
illustrated.  Cloth,  $3.50  net. 

JUST  OUT— INCLUDING  VACCINES  AND  SERUMS 

Dr.  Ward's  work  is  designed  to  be  of  service  to  the  man  in  general  practice. 
It  gives  you  the  exact  technic  for  obtaining  the  blood  for  examination,  the  making 
of  smears,  making  the  blood-count,  finding  coagulation  time,  etc.  Then  it  takes 
up  each  disease,  giving  you  the  synonyms,  definition,  nature,  general  pathology, 
etiology,  bearings  of  age  and  sex,  the  onset,  symptomatology  (discussing  each 
symptom  in  detail),  course  of  the  disease,  clinical  varieties,  complications,  diag- 
nosis, and  treatment  (drug,  diet,  rest,  vaccines  and  serums,  .r-ray,  operation,  etc.). 
There  is  a  special  chapter  devoted  to  the  medical  treatment  of  hemorrhage,  giving 
you  the  exact  doses  of  the  various  drugs  indicated,  and  the  methods  of  their 
administration,  the  serum  treatment,  transfusion,  etc.  Another  chapter  is  devoted 
to  the  value  of  blood  findings  in  surgical  diagnosis,  pointing  out  their  value  in 
differentiating  benign  from  malignant  growths,  infectious  from  other  diseases, 
appendicitis  from  typhoid  fever.  The  final  30  pages  are  given  over  to  a  summary 
of  the  blood  conditions  in  the  various  diseases,  arranged  alphabetically. 


Smith's  What  to  Eat  and  Why 

What  to  Eat  and  Why.  By  G.  CARROLL  SMITH,  M.D.,  Boston. 
I2mo  of  312  pages.  Cloth,  $2.50  net. 

FOR  THE  PRACTITIONER 

With  this  book  you  no  longer  need  send  your  patients  to  a  specialist  to  be 
dieted — you  will  be  able  to  prescribe  the  suitable  diet  yourself  just  as  you  do 
other  forms  of  therapy.  Dr.  Smith  gives  the  "why"  of  each  statement  he 
makes.  It  is  this  knowing  why  which  gives  you  confidence  in  the  book,  which 
makes  you  feel  that  Dr.  Smith  knows. 

Pennsylvania  Medical  Journal 

"All  through  this  book  Dr.  Smith  has  added  to  his  dietetic  hints  a  great  many  valuable  ones 
of  a  general  nature,  which  will  appeal  to  the  general  practitioner." 


Slade's  Physical  Examination  and  Diagnostic  Anatomy 

PHYSICAL  EXAMINATION  AND  DIAGNOSTIC  ANATOMY. — By  CHARLES  B.  SLADE,  M.D., 
Chief  of  Clinic  in  General  Medicine,  University  and  Bellevue  Hospital  Medical  College. 
I2mo  of  146  pages,  illustrated.  Cloth,  $1.25  net. 

"In  this  volume  is  contained  the  fundamental  methods  and  principles  of  physical  examination,  well 
illustrated,  largely  by  line  drawings.  The  book  is  to  be  strongly  recommended."— Boston  MedicaJ.  and 
Surgical  Journal. 


SAUNDERS'  BOOKS  ON 


Bastedo's   Materia    Medica 

Pharmacology,    Therapeutics,    Prescription    Writing 

Materia  Medica,  Pharmacology,  Therapeutics,  and  Prescription 
Writing.  By  W.  A.  BASTEDO,  PH.  D.,  M.  D.,  Associate  in  Pharma- 
cology and  Therapeutics  at  Columbia  University,  New  York.  Octavo 
of  602  pages,  illustrated.  Cloth,  $3.50  net. 

THREE  PRINTINGS  IN  SIX  MONTHS 

Dr.  Bastedo's  discussion  of  his  subject  is  very  complete.  As  an  illustration, 
take  the  pharmacologic  action  of  the  drug.  It  gives  you  the  antiseptic  action,  the 
local  action  on  the  skin,  mucous  membranes,  and  the  alimentary  tract  ;  where  the 
drug  is  obsorbed,  if  at  all — and  how  rapidly.  It  gives  you  the  systemic  action  on  the 
circulatory  organs,  respiratory  organs,  nervous  system,  and  sense  organs.  It  tells 
you  how  the  drug  is  changed  in  the  body.  It  gives  you  the  route  of  elimination 
and  in  what  form.  It  gives  you  the  action  on  the  kidneys,  bladder,  urethra,  skin, 
bowels,  lungs,  and  mammary  glands  during  elimination.  It  gives  you  the  after- 
effects. It  gives  you  the  unexpected — the  unusual — effects.  It  gives  you  the 
tolerance — habit  formation.  Could  any  discussion  be  more  complete,  more 
thorough  ? 

Boston  Medical  and  Surgical  Journal 

"  Its  aim  throughout  is  therapeutic  and  practical,  rather  than  theoretic  and  pharmacologic. 
The  text  is  illustrated  with  sixty  well-chosen  plates  and  cuts.  It  should  prove  a  useful  con- 
tribution to  the  text-book  literature  on  these  subjects." 


McKenzie  on  Exercise  in 
Education   and    Medicine 

Exercise  in  Education  and  Medicine.  By  R.  TAIT  MCKENZIE,  B.  Av 
M.  D.,  Professor  of.  Physical  Education  and  Director  of  the  Department, 
University  of  Pennsylvania.  Octavo  of  393  pages,  with  346  original 
illustrations.  Cloth,  $3.50  net. 

D.  A.  Sargeant,  M.  D.,  Director  of  Hemenway  Gymnasium,  Harvard  University. 

"It  cannot  fail  to  be  helpful  to  practitioners  in  medicine.  The  classification  of  athletic 
games  and  exercises  in  tabular  form  for  different  ages,  sexes,  and  occupations  is  the  work  of  an 
expert.  It  should  be  in  the  hands  of  every  physical  educator  and  medical  practitioner." 

Bonney's  Tuberculosis  second  Edition 

TUBERCULOSIS.     By  SHERMAN  G.  BONNEY,  M.  D.,  Professor  of  Medi- 
cine, Denver  and  Gross  College  of  Medicine.     Octavo  of  955  pages,  with 
243  illustrations.     Cloth,  $7.00  net;  Half  Morocco,  $8.50  net. 
Maryland  Medical  Journal 

"  Dr.  Bonney's  book  is  one  of  the  best  and  most  exact  works  on  tuberculosis,  in  all  its 
aspects,  that  has  yet  been  published." 


THE  PRACTICE   OF  MEDICINE 


Anders9 
Practice   of  Medicine 


A  Text-Book  of  the  Practice  of  Medicine.  By  JAMES  M.  ANDERS, 
M.  D.,  PH.  D.,  LL.  D.;  Professor  of  the  Practice  of  Medicine  and  of 
Clinical  Medicine,  Medico-Chirurgical  College,  Philadelphia.  Hand- 
some octavo,  1335  pages,  fully  illustrated.  Cloth,  $5.50  net;  Half 
Morocco,  $7.00  net. 

JUST  READY— THE  NEW  (llth)  EDITION 

The  success  of  this  work  is  no  doubt  due  to  the  extensive  consideration  given 
to  Diagnosis  and  Treatment,  under  Differential  Diagnosis  the  points  of  distinction 
of  simulating  diseases  being  presented  in  tabular  form.  In  this  new  edition 
Dr.  Anders  has  included  all  the  most  important  advances  in  medicine,  keeping 
the  book  within  bounds  by  a  judicious  elimination  of  obsolete  matter.  A  great 
many  articles  have  also  been  rewritten. 

Wm.  E.  Quine,  M.D.. 

Professor  of  Medicine  and  Clinical  Medicine,  College  of  Physicians  and  Surgeons,  Chicago. 
"  I  consider  Anders'  Practice  one  of  the  best  single- volume  works  before  the  profession  at 
this  time,  and  one  of  the  best  text-books  for  medical  students." 


DaCosta's  Physical  Diagnosis 

Physical  Diagnosis.  By  JOHN  C.  DACOSTA,  JR.,  M.  D.,  Associate 
in  Clinical  Medicine,  Jefferson  Medical  College,  Philadelphia.  Octavo 
of  557  pages,  with  225  original  illustrations.  Cloth,  $3.50  net. 

NEW  (2d)  EDITION 

Dr.  DaCosta's  work  is  a  thoroughly  new  and  original  one.  Every  method 
given  has  been  carefully  tested  and  proved  of  value  by  the  author  himself. 
Normal  physical  signs  are  explained  in  detail  in  order  to  aid  the  diagnostician  in 
determining  the  abnormal.  Both  direct  and  differential  diagnosis  are  emphasized. 
The  cardinal  methods  of  examination  are  supplemented  by  full  descriptions  of 
technic  and  the  clinical  utility  of  certain  instrumental  means  of  research. 
Dr.  Henry  L.  Eisner,  Professor  of  Medicine  at  Syracuse  University. 

"  I  have  reviewed  this  book,  and  am  thoroughly  convinced  that  it  is  one  of  the  best  ever 
written  on  this  subject.  In  every  way  I  find  it  a  superior  production." 


SAUNDERS*  BOOKS  ON 


Sahli's  Diagnostic  Methods 


A  Treatise  on  Diagnostic  Methods  of  Examination.  By  PROF. 
DR.  H.  SAHLI,  of  Bern.  Edited,  with  additions,  by  NATH'L  BOWDITCH 
POTTER,  M.  D.,  Assistant  Professor  of  Clinical  Medicine,  Columbia  Uni- 
versity (College  of  Physicians  and  Surgeons),  New  York.  Octavo  of 
1229  pages,  illustrated.  Cloth,  $6.50  net ;  Half  Morocco,  $8.00  net. 

THE  NEW  (2d)  EDITION,  ENLARGED  AND  RESET 

Dr.  Sahli's  great  work  is  a  practical  diagnosis,  written  and  edited  by  practical 
clinicians.  So  thorough  has  been  the  revision  for  this  edition  that  it  was  found 
necessary  practically  to  reset  the  entire  work.  Every  line  has  received  careful 
scrutiny,  adding  new  matter,  eliminating  the  old. 

Lewellys  F.  Barker,  M.  D. 

Professor  of  the  Principles  and  Practice  of  Medicine,  Johns  Hopkins  University 
"  I  am  delighted  with  it,  and  it  will  be  a  pleasure  to  recommend  it  to  our  students  in  the 
Johns  Hopkins  Medical  School." 

Friedenwald  and  Ruhrah  on  Diet 

Diet  in  Health  and  Disease.  By  JULIUS  FRIEDENWALD,  M.  D., 
Professor  of  Diseases  of  the  Stomach,  and  JOHN  RUHRAH,  M.  D.,  Pro- 
fessor of  Diseases  of  Children,  College  of  Physicians  and  Surgeons, 
Baltimore.  Octavo  of  857  pages.  Cloth,  $4.00  net. 

JUST  READY— THE  NEW  (4th)  EDITION 

This  new  edition  has  been  carefully  revised,  making  it  still  more  useful  than  the  two 
editions  previously  exhausted.  The  articles  on  milk  and  alcohol  have  been  rewritten,  additions 
made  to  those  on  tuberculosis,  the  salt-free  diet,  and  rectal  feeding,  and  several  tables  added, 
including  Winton's,  showing  the  composition  of  diabetic  foods. 

George  Dock,  M.  D. 

Professor  of  Theory  and  Practice  and  of  Clinical  Medicine,    Tulane   University. 
"  It  seems  to  me  that  you  have  prepared  the  most  valuable  work  of  the  kind  now  available. 
I  am  especially  glad  to  see  the  long  list  of  analyses  of  different  kinds  of  foods." 

Carter's  Diet  Lists  just  Ready 

DIET  LISTS  OF  THE  PRESBYTERIAN  HOSPITAL  OF  NEW  YORK  CITY. 
Compiled,  with  notes,  by  HERBERT  S.  CARTER,  M.  D.  i2mo  of  129 
pages.  Cloth,  $  i.  oo  net. 

Here  Dr.  Carter  has  compiled  all  the  diet  lists  for  the  various  diseases  and  for  conva- 
lescence as  prescribed  at  the  Presbyterian  Hospital.     Recipes  are  also  included. 


PRACTICE   OF  MEDICINE 


Kemp  on  Stomach, 
Intestines,  and  Pancreas 

Diseases  of  the  Stomach,  Intestines,  and  Pancreas.  By  ROBERT 
COLEMAN  KEMP,  M.  D.,  Professor  of  Gastro-intestinal  Diseases  at  the 
New  York  School  of  Clinical  Medicine.  Octavo  of  1021  pages,  with 
388  illustrations.  Cloth,  $6.50  net ;  Half  Morocco,  $8.00  net. 

NEW  (2d)  EDITION 

The  new  edition  of  Dr.  Kemp's  successful  work  appears  after  a  most  search- 
ing revision.  Several  new  subjects  have  been  introduced,  notably  chapters  on 
Colon  Bacillus  Infection  and  on  Diseases  of  the  Pancreas,  the  latter  article  being 
really  an  exhaustive  monograph,  covering  over  one  hundred  pages.  The  section 
on  Duodenal  Ulcer  has  been  entirely  rewritten.  Visceral  Displacements  are  given 
special  consideration,  in  every  case  giving  definite  indications  for  surgical  inter- 
vention when  deemed  advisable.  There  are  also  important  chapters  on  the  Intes- 
tinal Complications  of  Typhoid  Fever  and  on  Diver ticulitis. 

The  Therapeutic  Gazette 

"The  therapeutic  advice  which  is  given  is  excellent.  Methods  of  physical  and  clinical 
examination  are  adequately  and  correctly  described." 


Deaderick     on     Malaria 


Practical  Study  of  Malaria.  By  WILLIAM  H.  DEADERICK,  M.  D., 
Member  American  Society  of  Tropical  Medicine ;  Fellow  London 
Society  of  Tropical  Medicine  and  Hygiene.  Octavo  of  402  pages, 
illustrated.  Cloth,  $4.50  net;  Half  Morocco,  $6.00  net. 

Frank  A.  Jones,    M.  D.,  Memphis  Hospital  Medical  College. 

"We  have  been  waiting  for  many  years  for  such  a  work  written  by  a  man  who  sees  malaria 
in  all  its  forms  in  a  highly  malarious  climate." 


Two  Printings 
in  Six  Months 


Niles  on  Pellagra 

Pellagra.  By  GEORGE  M.  NILES,  M.  D.,  Professor  of  Gastro- 
enterology  and  Therapeutics,  Atlanta  School  of  Medicine.  Octavo  of 
253  pages,  illustrated.  Cloth,  $3.00  net. 

This  is  a  book  you  must  have  to  get  in  touch  with  the  latest  advances  con- 
cerning this  disease.  It  is  the  first  book  on  the  subject  by  an  American  author, 
and  the  first  in  any  language  adequately  covering  diagnosis  and  treatment. 
Pathology,  heretofore  an  echo  of  European  views  only,  is  here  presented  from  an 
American  point  of  view  as  well,  much  original  work  being  included.  The  clinical 
description  covers  the  manifestations  of  Pellagra  from  every  angle. 


I0  SAUNDERS'  BOOKS  ON 

AMERICAN   EDITION 

NOTHNAGEL'S  PRACTICE 

UNDER   THE    EDITORIAL    SUPERVISION   OF 

ALFRED   STENGEL,   M.D. 

Professor  of  Medicine  in  the  University  of  Pennsylvan'" 


Typhoid  and  Typhus  Fevers 

By  DR.  H.  CURSCHMANN,  of  Leipsic,  Edited,  with  additions,  by  WILLIAM 
OSLER,  M.  D.,  F.  R.  C.  P.,  Regius  Professor  of  Medicine,  Oxford  University, 
Oxford,  England.  Octavo  of  646  pages,  illustrated. 

Smallpox  including  Vaccination),  Varicella,  Cholera  Asiatica, 
Cholera  Nostras,  Erysipelas,  Erysipeloid,  Pertussis,  and 
Hay  Fever 

By  DR.  H.  IMMERMANN,  of  Rasle  ;  DR.  TH.  VON  JURGENSEN,  of  Tubingen  ; 
DR.  C.  LIEBERMEISTER,  of  Tubingen ;  DR.  H.  LENHARTZ,  of  Hamburg  ; 
and  DR.  G.  STICKER,  of  Giessen.  The  entire  volume  edited,  with  additions, 
by  SIR  J.  W.  MOORE,  M.  D.,  F.  R.  C.  P.  I.,  Professor  of  Practice,  Royal  Col- 
lege of  Surgeons,  Ireland.  Octavo  of  682  pages,  illustrated. 

Diphtheria,  Measles,  Scarlet  Fever,  and  Rotheln 

By  WILLIAM  P.  NORTHRUP,  M.  D.,  of  New  York,  and  DR.  TH.  VON  JUR- 
GENSEN, of  Tubingen.  The  entire  volume  edited,  with  additions,  by  WILLIAM 
P.  NORTHRUP,  M.  D.,  Professor  of  Pediatrics,  University  and  Bellevue  Hos- 
pital Medical  College,  New  York.  Octavo  of  672  pages,  illustrated,  including 
24  full-page  plates,  3  in  colors. 

Diseases  of  the  Bronchi,  Diseases  of  the  Pleura,  and  Inflam- 
mations of  the  Lungs 

By  DR.  F.  A.  HOFFMANN,  of  Leipsic ;  DR.  O.  ROSENBACH,  of  Berlin ;  and 
DR.  F.  AUFRECHT,  of  Magdeburg.  The  entire  volume  edited,  with  additions, 
by  JOHN  H.  MUSSER,  M.  D.,  University  of  Pennsylvania.  Octavo  of  1029 
pages,  illustrated,  including  7  full-page  colored  lithographic  plates. 

Diseases  of  the  Pancreas,  Suprarenals,  and  Liver 

By  DR.  L.  OSER,  of  Vienna  ;  DR.  E.  NEUSSER,  of  Vienna  ;  and  DRS.  H. 
QUINCKE  and  G.  HOPPE-SEYLER,  of  Kiel.  The  entire  volume  edited,  with 
additions,  by  REGINALD  H.  FRITZ,  A.  M.,  M.  D.,  Hersey  Professor  of  the 
Theory  and  Practice  of  Physic,  Harvard  University  ;  and  FREDERICK  A. 
PACKARD,  M.  D.,  Late  Physician  to  the  Pennsylvania  and  Children's  Hos- 
pitals, Philadelphia.  Octavo  of  918  pages,  illustrated. 

SOLD  SEPARATELY— PER  VOLUME:  CLOTH,  $5.00  NET;    HALF  MOROCCO,  $6.00  NET 


PRACTICE   OF  MEDICINE  II 

AMERICAN   EDITION 

NOTHNAGEL'S  PRACTICE 

Diseases  of  the  Stomach 

By  DR.  F.  RIEGEL,  of  Giessen.  Edited,  with  additions,  by  CHARLES  G. 
STOCKTON,  M.  D. ,  Professor  of  Medicine,  University  of  Buffalo.  Octavo  of 
835  pages,  with  29  text-cuts  and  6  full -page  plates. 

Diseases  of  the  Intestines  and  Peritoneum  Second  Edition 

By  DR.  HERMANN  NOTHNAGEL,  of  Vienna.  Edited,  with  additions,  by 
H.  D.  ROLLESTON,  M.  D.,  F.  R.  C.  P.,  Physician  to  St.  George's  Hospital, 
London.  Octavo  of  noo  pages,  illustrated. 

Tuberculosis  and  Acute  General  Miliary  Tuberculosis 

By  DR.  G.  CORNET,  of  Berlin.  Edited,  with  additions,  by  WALTER  B. 
JAMES,  M.  D,,  Professor  of  the  Practice  of  Medicine,  Columbia  University, 
New  York.  Octavo  of  806  pages. 

Diseases  Of  the  Blood   (Anemia,  Chlorosis,  Leukemia,  and  Pseudoleukemid) 

By  DR.  P.  EHRLICH,  of  Frankfort-on-the-Main  ;  DR.  A.  LAZARUS,  of  Char- 
lottenburg ;  DR.  K.  VON  NOORDEN,  of  Frankfort-on-the-Main ;  and  DR. 
FELIX  PINKUS,  of  Berlin.  The  entire  volume  edited,  with  additions,  by  ALFRED 
STENGEL,  M.D.,  Professor  of  Medicine,  University  of  Pennsylvania.  Octavo 
of  714  pages,  with  text-cuts  and  13  full-page  plates,  5  in  colors. 

Malarial  Diseases,  Influenza,  and  Dengue 

By  DR.  J.  MANNABERG,  of  Vienna,  and  DR.  O.  LEICHTENSTERN,  of  Cologne. 
The  entire  volume  edited,  with  additions,  by  RONALD  Ross,  F.  R.  C.  S.  (£NG.), 
F.  R.  S.,  Professor  of  Tropical  Medicine,  University  of  Liverpool  ;  J.  W.  W. 
STEPHENS,  M.  D.,  ,D.  P.  H.,  Walter  Myers  Lecturer  on  Tropical  Medicine, 
University  of  Liverpool ;  and  ALBERT  S.  GRUNBAUM,  F.  R.  C.  P. ,  Professor 
of  Experimental  Medicine,  University  of  Liverpool.  Octavo  of  769  pages, 
illustrated. 

Diseases  of  Kidneys  and  Spleen,  and  Hemorrhagic  Diatheses 

By  DR.  H.  SENATOR,  of  Berlin,  and  DR.  M.  LITTEN,  of  Berlin.  The  entire 
volume  edited,  with  additions,  by  JAMES  B.  HERRICK,  M.  D.,  Professor  of  the 
Practice  of  Medicine,  Rush  Medical  College.  Octuvo  of  815  pages,  illust. 

Diseases  of  the  Heart 

By  PROF.  DR.  TH.  VON  JURGENSEN,  of  Tubingen  ;  PROF.  DR.  L.  KREHL, 

of  Greifswaid  ;  and  PROF.  DR.   L.  VON  SCHROTTER,  of  Vienna.     Edited  by 

GEORGE  DOCK,  M.D.,  Professor  of  Theory  and  Practice  of  Medicine  and 

Clinical  Medicine,  Tulane  University.     Octavo,  848  pages,  illustrated. 

SOLD  SEPARATELY— PER  VOLUME:    CLOTH,  $5.00  NET  ;    HALF  MOROCCO,  $6.00  NET 

Goepp's    State    Board    Questions 

JUST  READY— NEW  (3d)  EDITION 

State  Board  Questions  and  Answers.  By  R.  MAX  GOEPP,  M.D., 
Professor  of  Clinical  Medicine,  Philadelphia  Polyclinic.  Octavo  of  7 1 5 
pages.  Cloth,  $4.00  net ;  Half  Morocco,  $5.50  net 

Pennsylvania  Medical  Journal 

"  Nothing  has  been  printed  which  is  so  admirably  adapted  as  a  guide  and  self-quiz  for  those 
intending  to  take  State  Board  Examinations." 


12  SAUNDERS'    BOOKS   ON 

Stevens'  Therapeutics  New  (sth)  Edition 

A  TEXT-BOOK  OF  MODERN  MATERIA  MEDICA  AND  THERAPEUTICS. 
By  A.  A.  STEVENS,  A.  M.,  M.  D.,  Lecturer  on  Physical  Diagnosis  in 
the  University  of  Pennsylvania.  Octavo  of  675  pages.  Cloth,  $3.50  net. 

Dr.  Stevens'  Therapeutics  is  one  of  the  most  successful  works  on  the 
subject  ever  published.  In  this  new  edition  the  work  has  undergone  a 
very  thorough  revision,  and  now  represents  the  very  latest  advances. 

The  Medical  Record,  New  York 

"  Among  the  numerous  treatises  on  this  most  important  branch  of  medical  practice, 
this  by  Dr.  Stevens  has  ranked  with  the  best." 

Butler's  Materia  Medic  a  New  (6th)  Edition 

A  TEXT-BOOK  OF  MATERIA  MEDICA,  THERAPEUTICS,  AND  PHARMA- 
COLOGY. By  GEORGE  F.  BUTLER,  PH.  G.,  M.  D.,  Professor  and  Head 
of  the  Department  of  Therapeutics  and  Professor  of  Preventive  and 
Clinical  Medicine,  Chicago  College  of  Medicine  and  Surgery,  Medical 
Department  Valpariso  University.  Octavo  of  702  pages,  illustrated. 
Cloth,  $4.00  net;  Half  Morocco,  $5.50  net. 

For  this  sixth  edition  Dr.  Butler  has  entirely  remodeled  his  work,  a  great 
part  having  been  rewritten.  All  obsolete  matter  has  been  eliminated,  and 
special  attention  has  been  given  to  the  toxicologic  and  therapeutic  effects 
of  the  newer  compounds. 

Medical  Record,  New  York 

"  Nothing  has  been  omitted  by  the  author  which,  in  his  judgment,  would  add  to  the 
completeness  of  the  text." 

'"  •  * 

Sollmann's  Pharmacology  New  (2d  edition 

A  TEXT-BOOK  OF  PHARMACOLOGY.  By  TORALD  SOLLMANN,  M.  D., 
Professor  of  Pharmacology  and  Materia  Medica,  Western  Reserve  Uni- 
versity. Octavo  of  1070  pages,  illustrated.  Cloth,  $4.00  net. 

The  author  bases  the  study  of  therapeutics  on  systematic  knowledge  of 
the  nature  and  properties  of  drugs,  and  thus  brings  out  forcibly  the  intimate 
relation  between  pharmacology  and  practical  medicine. 

J.  F.  Fotheringham,  M.  D.,    Trinity  Medical  College,    Toronto. 

"  The  work  certainly  occupies  ground  not  covered  in  so  concise,  useful,  and  scientific  a 
manner  by  any  other  text  I  have  read  on  the  subjects  embraced." 

Arny's  Pharmacy 

PRINCIPLES  OF  PHARMACY.  By  HENRY  V.  ARNY,  PK.  G.,  PH.  D., 
Columbia  University,  New  York.  Octavo  of  1175  pages,  with  246  illus- 
trations. Cloth,  $5.00  net. 

George  Reimann,  Ph.  G.,  Secretary  of  the  New   York  State  Board  of  Pharmacy. 

"  I  would  say  that  the  book  is  certainly  a  great  help  to  the  student,  and  I  think  it  ought 
to  be  in  the  hands  of  every  person  who  is  contemplating  the  study  of  pharmacy." 


THERAPEUTICS  AND  MATERIA  MEDICA  13 

Hinsdale's  Hydrotherapy 

Hydrotherapy :  A  Treatise  on  Hydrotherapy  in  General;  Its 
Application  to  Special  Affections ;  the  Technic  or  Processes  Employed, 
and  a  Brief  Chapter  on  the  Use  of  Waters  Internally.  By  GUY  H INS- 
DALE,  M.  D.,  Fellow  Royal  Society  of  Medicine  of  Great  Britain. 
Octavo  of  466  pages,  illustrated.  Cloth,  $3.50  net. 

INCLUDING  CROUNOTHERAPY 

The  treatment  of  disease  by  hydrotherapeutic  measures  has  assumed  such  an 
important  place  in  Medical  practice  that  a  good,  practical  work  on  the  subject 
is  an  essential  in  every  practitioner's  armamentarium.  This  new  work  supplies 
all  needs.  It  describes  fully  the  various  kinds  of  baths,  douches,  sprays  ;  the 
application  of  heat  and  cold  ;  the  internal  use  of  mineral  waters  and  all  other 
procedures  included  under  hydrotherapeutic  measures. 

The  Medical  Record 

"  We  cannot  conceive  of  a  work  more  useful  to  the  general  practitioner  than  this,  nor  one 
to  which  he  would  resort  more  frequently  for  reference  and  guidance  in  his  daily  work." 


Kelly's  Cyclopedia  of  Ameri- 
can Medical  Biography 

Cyclopedia  of  American  Medical  Biography.  By  HOWARD  A. 
KELLY,  M.  D.,  Johns  Hopkins  University.  Two  octavos,  averaging  525 
pages  each,  with  portraits.  Per  set:  Cloth,  $10.00  net;  Half  Morocco, 

$13.00  net. 

IN  TWO  VOLUMES 

Dr.  Kelly,  in  these  two  handsome  volumes,  presents  concise,  yet  complete, 
biographies  of  those  men  and  women  who  have  contributed  noteworthily  to  the 
advancement  of  medicine  in  America.  Dr.  Kelly's  reputation  for  painstaking 
care  assures  accuracy  of  statement.  There  are  about  one  thousand  biographies 
included. 

Swan's  Prescription-writing  and  Formulary 

PRESCRIPTION-WRITING  AND  FORMULARY.  By  JOHN  M.  SWAN,  M.  D.,  formerly 
Director  Glen  Springs  Sanitarium,  Watkins,  N.  Y.  l6mo  of  185  pages.  Flexible 
leather,  $1.25  net. 


Stewart's  Pocket  Therapeutics  and  Dose-book 

POCKET  THERAPEUTICS  AND  DOSE-BOOK.    By  MORSE  STEWART,  JR.,  M.D.    32mo 
of  263  pages.     Cloth,  #1.00  net. 


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Illustrated    Dictionary 


New  (7th)  Edition—  5000  Sold  in  Two  Months 

The  American  Illustrated  Medical  Dictionary  __  By  W.  A.  NEW- 

MAN BORLAND,  M.  D.,  Editor  of  "The  American  Pocket  Medical  Dic- 
tionary." Large  octavo  of  1107  pages,  bound  in  full  flexible  leather. 
Price,  $4.50  net;  with  thumb  index,  $5.00  net. 

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Howard  A.  Kelly,  M.S).t  Professor  of  Gynecologic  Surgery,  Johns  Hopkins  University. 

"  Dr.  Dorland's  dictionary  is  admirable.     It  is  so  well  gotten  up  and  of  such  convenient 
size.     No  errors  have  been  found  in  my  use  of  it." 


Thornton's  Dose-Book.  New  (4th)  Edition 

DOSE- BOOK  AND  MANUAL  OF  PRESCRIPTION-WRITING.  By  E.  Q.  THORNTON,  M.D., 
Assistant  Professor  of  Materia  Medica,  Jefferson  Medical  College,  Philadelphia.  Post- 
octavo,  410  pages,  illustrated.  Flexible  leather,  $2.00  net. 

"  I  will  be  able  to  make  considerable  use  of  that  part  of  its  contents  relating  to  the  correct 
terminology  as  used  in  prescription-writing,  and  it  will  afford  me  much  pleasure  to  recom- 
mend the  book  to  my  classes,  who  often  fail  to  find  this  information  in  their  other  text- 
books."— C.  H.  MILLER,  M.  ^..Professor  of  Pharmacology,  Northwestern  University  Medi- 
cal School. 

Lusk  on  Nutrition  New  (2d)  Edition 

ELEMENTS  OF  THE  SCIENCE  OF  NUTRITION.  By  GRAHAM  LUSK,  PH.  D.,  Professor 
of  Physiology  in  Cornell  University  Medical  School.  Octavo  of  402  pages.  Cloth, 
$3.00  net. 

"  I  shall  recommend  it  highly.  It  is  a  comfort  to  have  such  a  discussion  of  the  subject." 
— LEWELLYS  F.  BARKER,  M.  D.,  Johns  Hopkins  University. 

Camac's  "Epoch-making  Contributions" 

EPOCH-MAKING  CONTRIBUTIONS  IN  MEDICINE  AND  SURGERY.  Collected  and 
arranged  by  C.  N.  B.  CAM  AC,  M.  D.,  of  New  York  City.  Octavo  of  450  pages,  illus- 
trated. Artistically  bound,  $4.00  net. 

"  Dr.  Camac  has  provided  us  with  a  most  interesting  aggregation  of  classical  essays^ 
We  hope  that  members  of  the  profession  will  show  their  appreciation  of  his  endeavors."— 
THERAPEUTIC  GAZETTE. 


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The  American  Pocket  Medical  Dictionary  New  (8th)  Edition 

THE  AMERICAN  POCKET  MEDICAL  DICTIONARY.  Edited  by  W.  A.  NEWMAN  DOR- 
LAND,  M.  D.,  Editor  "  American  Illustrated  Medical  Dictionary."  677  pages.  Flexible 
leather,  with  gold  edges,  $1.00  net;  with  thumb  index,  $1.25  net. 

Pusey  and  Caldwell  on  X-Rays  Second  Edition 

THE  PRACTICAL  APPLICATION  OF  THE  RONTGEN  RAYS  IN  THERAPEUTICS  AND 
DIAGNOSIS.  By  WILLIAM  ALLEN  PUSEY,  A.  M.,  M.  D.,  Professor  of  Dermatology  in 
the  University  of  Illinois  ;  and  EUGENE  W.  CALDWELL,  B.  S.,  Director  of  the  Edward 
N.  Gibbs  X-Ray  Memorial  Laboratory  of  the  University  and  Bellevue  Hospital  Medical 
College,  New  York.  Octavo  of  625  pages,  "with  200  illustrations.  Cloth,  $5.00  net; 
Half  Morocco,  $6.50  net. 

Cohen   and    Eshner's    Diagnosis.      Second  Revised  Edition 

ESSENTIALS  OF  DIAGNOSIS.  By  S.  SOLIS-COHEN,  M.  D.,  Senior  Assistant  Professor 
in  Clinical  Medicine,  Jefferson  Medical  College,  Phila.  ;  and  A.  A.  ESHNER,  M.  D., 
Professor  of  Clinical  Medicine,  Philadelphia  Polyclinic.  Post-octavo,  382  pages  ;  55 
illustrations.  Cloth,  $1.00  net.  In  Saunders1  Question-  Contend  Series. 

Morris'  Materia  Medica  and  Therapeutics.  New  (7th)  Edition 

ESSENTIALS  OF  MATERIA  MEDICA,  THERAPEUTICS,  AND  PRESCRIPTION-WRITING 
By  HENRY  MORRIS,  M.  D.,  late  Demonstrator  of  Therapeutics,  Jefferson  Medical 
College,  Phila.  Revised  by  W.  A.  BASTEDO,  M.  D.,  Instructor  in  Materia  Medica  and 
Pharmacology  at  Columbia  University.  1  2mo,  300  pages.  Cloth,  #l.oo  net.  In  Saunders' 
Question-  Commend  Series. 

Williams'  Practice  of  Medicine 

ESSENTIALS  OF  THE  PRACTICE  OF  MEDICINE.  By  W.  R.  WILLIAMS,  M.D., 
formerly  Instructor  in  Medicine  and  Lecturer  on  Hygiene,  Cornell  University  ;  and 
Tutor  in  Therapeutics,  Columbia  University,  N.  Y.  I2mo  of  456  pages,  illustrated. 
In  Saunders1  Question-  Compend  Series.  Double  number,  $1.75  net. 

Todd's  Clinical  Diagnosis  The  Ncw  (2d)  Edition 

A  MANUAL  OF  CLINICAL  DIAGNOSIS.     By  JAMES  CAMPBELL  TODD,  M.  D  ,  Professor 
of  Pathology,  University  of  Colorado.     I2mo  of  469  pages,  with   164  text-il 
and  10  colored  plates.     Cloth,  $2.25  net. 

Bridge  on  Tuberculosis 

TUBERCULOSIS.      By  NORMAN   BRIDGE,   A.  M      M.  D 

in  Rush  Medical  College.     I2mo  of  302  pages,  illustrated.    Cloth, 

Oertel  on  Bright'*  Disease  illustrated 


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Jakob  and  Eshner's  Internal  Medicine  and  Diagnosis 

ATLAS  AND  EPITOME  OF  INTERNAL  MEDICINE  AND  CLINICAL  DIAGNOSIS.  By  DR. 
CHR.  JAKOB,  of  Erlangen.  Edited,  with  additions,  by  A.  A.  ESHNER,  M.  D.,  Pro- 
fessor of  Clinical  Medicine,  Philadelphia  Polyclinic.  With  182  colored  figures  on 
68  plates,  64  text-illustrations,  259  pages  of  text.  Cloth,  $3.00  net.  In  Saunders1 
Hand-Atlas  Series. 


Lockwood's  Practice  of  Medicine.  »  , 

Revised  and  Enlarged 

A  MANUAL  OF  THE  PRACTICE  OF  MEDICINE.  By  GEO.  ROE  LOCKWOOD,  M.  D., 
Attending  Physician  to  the  Bellevue  Hospital,  New  York  City.  Octavo,  847  pages, 
with  79  illustrations  in  the  text  and  22  full-page  plates.  Cloth,  $4.00  net. 

Barton  and  Wells'  Medical  Thesaurus 

A  THESAURUS  OF  MEDICAL  WORDS  AND  PHRASES.  By  W.  M.  BARTON,  M.  D.,  and 
W.  A.  WELLS,  M.  D.,  of  Georgetown  University,  Washington,  D.  C.  I2mo  of  535 
pages.  Flexible  leather,  $2.50  net;  thumb  indexed,  $3.00  net. 

Jelliffe's    Pharmacognosy 

AN  INTRODUCTION  TO  PHARMACOGNOSY.  By  SMITH  ELY  JELLIFFE,  PH.  D.,  M.  D.., 
of  Columbia  University.  Octavo,  illustrated.  Cloth,  $2.50  net. 

Stevens'  Practice  of  Medicine  New  (9th)  Edition 

A  MANUAL  OF  THE  PRACTICE  OF  MEDICINE.     By  A.  A.  STEVENS,  A.  M.,  M.  D., 

Professor  of   Pathology,   Woman's   Medical   College,    Phila.  Specially  intended  for 

students  preparing  for  graduation  and  hospital  examinations.  Post-octavo,  573  pages, 
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